CN1656067A - new salt - Google Patents

Abstract

The present invention provides pharmaceutically acceptable acid addition salts of compounds of formula wherein R¹Represents C substituted by one or more fluoro substituents_1－2An alkyl group; r²Represents C_1－2An alkyl group; and n represents 0, 1 or 2, which salts are useful as prodrugs of competitive inhibitors of trypsin-like proteases, such as thrombin, and thus, are particularly useful in the treatment of conditions in which inhibition of thrombin is required (e.g. thrombosis) or as anticoagulants.

Description

new salt

field of invention

The present invention relates to novel salts of compounds which inhibit thrombin after administration to a mammalian patient, pharmaceutical compositions containing said salts, and processes for the preparation of said salts.

Background of the Invention and Prior Art

When formulating pharmaceutical compositions, it is important that the drug be in a form that can be easily handled and processed. This is important not only from the point of view of obtaining a commercially viable method of preparation, but also from the point of view of the subsequent preparation of pharmaceutical formulations comprising the active compound.

Furthermore, when preparing pharmaceutical compositions, it is also important to provide a reliable, reproducible and constant plasma drug concentration profile after administration.

The chemical stability, solid state stability and "shelf life" of the active ingredient are also very important factors. Drugs and drug-containing compositions should preferably be capable of being efficiently stored over long periods of time without exhibiting significant changes in the physicochemical characteristics of the active ingredient, such as its chemical composition, density, hygroscopicity and solubility.

Furthermore, it is also important to be able to provide the drug in a form that is as chemically pure as possible.

It will be appreciated by those skilled in the art that, in general, if a drug is readily available in a stable form, such as a stable crystalline form, this provides advantages of ease of handling, preparation of suitable pharmaceutical formulations, and more reliable solubility characteristics.

International Patent Application PCT/SE01/02657 (WO 02/44145, earliest priority date December 1, 2000, filed November 30, 2001, published June 6, 2002) discloses compounds, these The compounds are competitive inhibitors of trypsin-like proteases, such as thrombin, or are metabolized to compounds that are such inhibitors. Among them, the following three compounds are specifically disclosed:

(a) Ph(3-C1)(5-OCHF ₂ )-(R)CH(OH)C(O)-(S)Aze-Pab(OMe):

This compound is hereinafter referred to as Compound A;

(b) Ph(3-C1)(5-OCHF ₂ )-(R)CH(OH)C(O)-(S)Aze-Pab(2,6-diF)(OMe):

This compound is hereinafter referred to as Compound B; and

(c) Ph(3-C1)(5- _OCH2CH2F )-(R)CH(OH)C(O)-(S) _Aze -Pab(OMe):

This compound is referred to as Compound C hereinafter.

Abbreviations are listed at the end of this specification.

Methoxyamidine compounds A, B and C are metabolized after oral and/or parenteral administration to the corresponding free amidine compounds which have been found to be potent thrombin inhibitors.

Methods for the synthesis of compounds A, B and C are described in Examples 12, 40 and 22, respectively, of International Patent Application PCT/SE01/02657.

No specific pharmaceutically acceptable salts of compounds A, B or C are disclosed in PCT/SE01/02657. Furthermore, no information is provided on how to prepare compounds A, B or C in crystalline form, in particular salts thereof.

invention disclosure

According to a first aspect of the present invention, the present invention provides a pharmaceutically acceptable acid addition salt of a compound of formula I,

in

R ¹ represents C _1-2 alkyl substituted by one or more fluorine substituents;

R ² represents C _1-2 alkyl; and

n stands for 0, 1 or 2;

Said salts are hereinafter referred to as "compounds of the invention".

The compounds of the present invention may be in the form of solvates, hydrates, mixed solvates/hydrates, or preferably ansolvates, eg, anhydrates. The solvent may be one or more organic solvents such as lower (e.g. C _1-4 ) alkyl alcohols (e.g. methanol, ethanol or isopropanol), ketones (e.g. acetone), esters (e.g. ethyl acetate) or mixtures thereof . In addition, tautomers of the compounds of the present invention are also included.

Preferred acid addition salts include inorganic acid addition salts such as those of sulfuric acid, nitric acid, phosphoric acid and hydrohalic acids such as hydrobromic acid and hydrochloric acid. More preferred acid addition salts include those of organic acids, such as the addition salts of the following acids: dimethylphosphoric acid; saccharic acid; cyclamic acid; carboxylic acids (e.g. maleic acid, fumaric acid, Aspartic acid, succinic acid, malonic acid, acetic acid, benzoic acid, terephthalic acid, hippuric acid, 1-hydroxy-2-naphthoic acid, pamoic acid, hydroxybenzoic acid, etc.); hydroxy acids (such as salicylic acid acid, tartaric acid, citric acid, malic acid (including L-(-)-malic acid and D,L-malic acid), gluconic acid (including D-gluconic acid), glycolic acid, ascorbic acid, lactic acid, etc.); amino acids (such as glutamic acid (including D-glutamic acid, L-glutamic acid and D, L-glutamic acid), arginine (including L-arginine), lysine (including L-lysine and L-lysine hydrochloride), glycine, etc.); and especially sulfonic acids (such as 1,2-ethanedisulfonic acid, camphorsulfonic acid (including 1S-(+)-10-camphorsulfonic acid and (+ /-)-camphorsulfonic acid), ethanesulfonic acid, propanesulfonic acid (including n-propanesulfonic acid), butanesulfonic acid, pentanesulfonic acid, toluenesulfonic acid, methanesulfonic acid, p-xylenesulfonic acid, 2- Mesitylenesulfonic acid, naphthalenesulfonic acid (including 1,5-naphthalenesulfonic acid and 1-naphthalenesulfonic acid), benzenesulfonic acid, hydroxybenzenesulfonic acid, 2-hydroxyethanesulfonic acid, 3-hydroxyethanesulfonic acid, etc.) .

Particularly preferred salts include those formed with C _1-6 (e.g. C _1-4 ) alkanesulfonic acids such as ethanesulfonic acid and propanesulfonic acid (e.g. n-propanesulfonic acid) and optionally substituted (e.g. substituted by one or more C _1-2 alkyl) arylsulfonic acids such as benzenesulfonic acid.

Also particularly preferred salts include salts with C _1-6 (eg C _1-4 ) alkanesulfonic acids such as ethanesulfonic acid and propanesulfonic acid (eg n-propanesulfonic acid), optionally substituted ( For example arylsulfonic acids substituted by one or more C _1-2 alkyl groups) such as benzenesulfonic acid, and optionally substituted (for example substituted by one or more C _1-2 alkyl groups) aryl disulfonic acids Acids such as 1,5-naphthalenedisulfonic acid (and hemi-1,5-naphthalenedisulfonic acid).

Preferred compounds of formula I include those defined as follows:

R ¹ represents -OCHF ₂ or -OCH ₂ CH ₂ F;

R ² represents a methyl group;

n stands for 0 or 2.

More preferred compounds of formula I include those defined as follows: n represents 0, or n represents ₂ , thus providing Two fluorine atoms in two ortho positions).

Particularly preferred compounds of formula I include compound B, compound C and especially compound A.

The compounds of the present invention may be prepared by techniques which may include adding an appropriate amount of the corresponding acid to a compound of formula I in free base form, for example as described below; converting one salt into another (when the corresponding acid pKa values and the solubility of the respective salts are different); and ion-pair chromatography.

According to another aspect of the present invention, the present invention provides a process for preparing a compound of the present invention, comprising adding an acid to a compound of formula I.

A suitable stoichiometric ratio of acid to free base is 0.25:1.5-3.0:1, for example 0.45:1.25-1.25:1, including 0.50:1-1:1.

Compounds of formula I can be prepared by the following methods (relevant information incorporated herein is also referred to International Patent Application PCT/SE01/02657 (WO 02/44145, the earliest priority date is December 1, 2000, November 30, 2001) Submitted, published June 6, 2002)):

(i) compound of formula II

where ^R1 is as defined above,

Coupled with a compound of formula III,

where n and ^R2 are as defined above,

For example, the reaction is performed in the presence of a coupling reagent (such as oxalyl chloride, EDC, DCC, HBTU, HATU, PyBOP or TBTU in DMF), a suitable base (such as pyridine, DMAP, TEA, 2,4,6-trimethyl pyridine or DIPEA) and a suitable organic solvent (such as dichloromethane, acetonitrile, EtOAc or DMF);

(ii) compound of formula IV

where ^R1 is as defined above,

Coupled with a compound of formula V,

where n and ^R2 are as defined above,

The reaction is carried out, for example, under the conditions described in method (i) above; or

(iii) Derivatives of protected compounds corresponding to compounds of formula I, but present in place of the group OR ² (i.e. the corresponding free amidine compounds), said protected derivatives being e.g. compounds of formula VI or its tautomer

wherein R ^a represents for example -CH ₂ CH ₂ -Si(CH ₃ ) ₃ or benzyl, and R ¹ and n are as defined above, reacted with a compound of formula VII or an acid addition salt thereof,

R ² ONH ₂ VII

where ^R2 is as defined above,

For example, the reaction is carried out in the presence of a suitable organic solvent (such as THF, CH ₃ CN, DMF or DMSO) at room temperature to reflux temperature,

The -C(O) ^ORa group is then removed under conditions known to those skilled in the art (eg by reaction with QF or TFA (eg as described below)).

Compounds of formula II may be prepared using known and/or standard techniques.

For example, a compound of formula II can be prepared by converting an aldehyde of formula VIII

where ^R1 is as defined above,

Reacts with the following compounds:

(a) react with formula IX compound

                                        

wherein R″ represents H or (CH ₃ ) ₃ Si,

For example, the reaction is carried out at room temperature or elevated temperature (e.g. below 100°C) in the presence of a suitable organic solvent (e.g. chloroform or dichloromethane) and, if desired, in a suitable base (e.g. TEA) and/or a suitable in the presence of a catalyst system (e.g. benzyl ammonium chloride or zinc iodide, or using a chiral catalyst, e.g. as described in Chem. Rev., (1999) 99, 3649), and then under conditions well known to those skilled in the art Hydrolysis (for example as described below);

(b) reaction with NaCN or KCN, for example, the reaction is carried out in the presence of NaHSO ₃ and water, followed by hydrolysis;

(c) reaction with chloroform, e.g. the reaction is at elevated temperature (e.g. above room temperature but below 100°C) in the presence of a suitable organic solvent (e.g. chloroform) and if desired in a suitable catalyst system (e.g. chlorinated benzyl ammonium), followed by hydrolysis;

(d) react with the compound of formula X

where M represents Mg or Li,

followed by oxidative cleavage (e.g. ozonolysis or osmium or ruthenium catalyzed) under conditions well known to those skilled in the art; or

(e) Reaction with tris(methylthio)methane under conditions well known to those skilled in the art, followed by hydrolysis in the presence of eg HgO and _HBF4 .

Alternatively, compounds of formula II may be prepared by oxidizing compounds of formula XI or derivatives thereof optionally protected at secondary hydroxyl groups

where ^R1 is as defined above,

The oxidation is in the presence of a suitable oxidizing agent, such as a combination of a suitable free radical oxidizing agent such as TEMPO and a suitable hypochlorite such as sodium hypochlorite, under conditions known to those skilled in the art, such as at -10°C to room temperature, in a suitable solvent (e.g. water, acetone or a mixture thereof), a suitable salt (e.g. an alkali metal halide such as potassium bromide) and a suitable base (e.g. an alkali metal carbonate or bicarbonate such as sodium bicarbonate ) in the presence of.

In forming compounds of formula II, those skilled in the art will appreciate that the desired enantiomeric form may be prepared by conventional enantiomeric separation techniques, for example by enantiospecific derivatization steps. This can be achieved, for example, by enzymatic methods. Such enzymatic methods include, for example, at room temperature to reflux temperature (e.g. at 45-65°C) in a suitable enzyme (e.g. Lipase PS Amano), a suitable ester (e.g. vinyl acetate) and a suitable solvent (e.g. methyl tertiary α-OH transesterification in the presence of butyl ether). The derivatized isomer can then be separated from the unreacted isomer by conventional separation techniques such as chromatography.

Groups added to compounds of formula II during such derivatization steps may be removed prior to any further reaction in the synthesis of compounds of formula I or at any subsequent step. Additional groups can be removed using conventional techniques (e.g. for esters of α-OH, hydrolysis under conditions known to those skilled in the art (e.g. from room temperature to reflux temperature in a suitable base (e.g. NaOH) and a suitable solvent ( For example in the presence of MeOH, water or mixtures thereof))).

Compounds of formula III can be prepared by coupling (S)-azetidine-2-carboxylic acid with compounds of formula V as defined above under conditions similar to those described for the preparation of compounds of formula I next.

Compounds of formula IV can be prepared by coupling compounds of formula II as defined above with (S)-azetidine-2-carboxylic acid under conditions similar to those described for the preparation of compounds of formula I next.

Compounds of formula VI can be prepared by reacting compounds of formula II as defined above with compounds of formula XII,

where n and Ra ^are as defined above,

For example, the reaction is carried out under conditions similar to those described above for the synthesis of compounds of formula I.

Alternatively, a compound of formula VI can be prepared by reacting a compound corresponding to a compound of formula I, but in which an H atom is present in place of the group ^—OR (i.e. the corresponding free amidine compound) with a compound of formula XIII,

L ¹ COOR ^a XIII

wherein ^L represents a suitable leaving group such as halogen or nitrophenyl (eg 4-nitrophenyl), and R ^is as defined above,

For example, the reaction is carried out at about room temperature in the presence of a suitable base (eg NaOH, eg aqueous NaOH) and a suitable organic solvent (eg dichloromethane).

Compounds of formula VIII may be prepared using known and/or standard techniques. For example, they can be prepared by:

(i) metallating a compound of formula XIV (where the metal may be, for example, an alkali metal such as Li, or preferably a divalent metal such as Mg),

Wherein Hal represents a halogen atom selected from Cl, Br and I, and ^R is as defined above,

Then react with a suitable formyl source (eg N,N-dimethylformamide) under conditions such as those described below;

(ii) compound of formula XV

where ^R1 is as defined above,

Reduction in the presence of a suitable reducing agent (eg DIBAL-H);

(iii) compound of formula XVI

where ^R1 is as defined above,

Oxidation in the presence of a suitable oxidizing agent such as _MnO2 , pyridinium chlorochromate, DMSO in combination with oxalyl chloride or _SO3 pyridine complex in DMSO.

Compounds of formula XII can be prepared by reacting (S)-azetidine-2-carboxylic acid with compounds of formula XVII

where n and Ra ^are as defined above,

For example, the reaction is carried out under conditions similar to those described above for the synthesis of compounds of formula I.

Formulas V, VII, IX, X, XI, XIII, XIV, XV, XVI, XVII and (S)-azetidine-2-carboxylic acid are commercially available, known in the literature, or can be obtained by analogy They are obtained by the methods described herein or by conventional synthetic methods, according to standard techniques, from readily available starting materials using suitable reagents and reaction conditions. The free amidine equivalents of compounds of formula I can be prepared in a manner analogous to that described herein for the preparation of compounds of formula I.

We have found that some of the compounds of the present invention have the advantage that they can be prepared in crystalline form.

According to another aspect of the invention, the present invention provides a compound of the invention in substantially crystalline form.

Although we have found that compounds of the invention that are greater than 80% crystalline can be prepared, "substantially crystalline" includes greater than 20%, preferably greater than 30%, more preferably greater than 40% (e.g. greater than 50%, 60%, 70%, 80% Or 90%) crystallization.

According to another aspect of the present invention, the present invention also provides a compound of the present invention in partially crystalline form. "Partially crystalline" includes 5% or 5% to 20% crystalline.

Crystallinity (%) can be measured by those skilled in the art using X-ray powder diffraction (XRPD). Other techniques such as solid state NMR, FT-IR, Raman spectroscopy, differential scanning calorimetry (DSC) and microcalorimetry can also be used.

Compounds of the invention, especially crystalline compounds of the invention, may have improved stability compared to the compounds disclosed in PCT/SE01/02657.

The term "stability" as defined herein includes chemical stability and solid state stability.

"Chemical stability" means that the compound of the present invention can be administered in an isolated form or in a formulation wherein the compound of the present invention is mixed with a pharmaceutically acceptable carrier, diluent or adjuvant (for example, in oral dosage forms such as tablets, capsules, etc. ) stored under standard storage conditions without significant chemical degradation or decomposition.

"Solid state stability" means that the compound of the present invention can be in the form of an isolated solid or a solid preparation in which the compound of the present invention is mixed with a pharmaceutically acceptable carrier, diluent or adjuvant (e.g. in oral dosage forms such as tablets, capsules, etc.) etc.) without significant solid-state transformations (eg, crystallization, recrystallization, solid-state phase transition, hydration, dehydration, solvation or desolvation) under standard storage conditions.

Examples of "standard storage conditions" include temperatures from -80°C to +50°C (preferably 0-40°C, more preferably room temperature such as 15-30°C) over a long period of time (i.e. greater than or equal to 6 months), 0.1- A pressure of 2 bar (preferably atmospheric pressure), a relative humidity of 5%-95% (preferably 10%-60%), and/or exposure to UV/visible light of 460 lux. Under such conditions, the compounds of the invention may undergo less than 15%, more preferably less than 10% and especially less than 5% chemical degradation/decomposition or solid state transformation. Those skilled in the art will appreciate that the above upper and lower limits for temperature, pressure and relative humidity represent extremes of standard storage conditions, and during standard storage some combinations of these extreme conditions (e.g. a temperature of 50°C and a pressure of 0.1 bar) may will not experience.

Preferred compounds of the invention which can be obtained in crystalline form include salts with C _1-6 (eg C _1-6 such as C _2-4 ) alkanesulfonic acids such as ethanesulfonic acid and propanesulfonic acid (eg n-propanesulfonic acid) and optionally substituted arylsulfonic acids such as benzenesulfonic acid.

The salts of Compounds A, B and C may be crystallized with or without solvent systems (for example crystallization may occur from the melt, under supercritical conditions or by sublimation). However, we prefer to carry out the crystallization from a suitable solvent system.

Suitable solvent systems useful in the crystallization process may be heterogeneous or homogeneous and thus may comprise one or more organic solvents such as lower alkyl acetates (e.g. linear or branched C _1-6 alkyl acetates such as ethyl acetate, isopropyl acetate and butyl acetate); lower (for example linear or branched C _1-6 alkyl) alkyl alcohols such as hexan-1-ol, 3-methylbutan-1-ol, Pentan-1-ol, Pentan-2-ol, 4-methyl-2-pentanol and 2-methyl-1-propanol, methanol, ethanol, n-propanol, isopropanol and butanol (e.g. n-butanol) ; aliphatic hydrocarbons (such as straight-chain or branched C _5-8 alkanes such as n-pentane, n-heptane and isooctane); aromatic hydrocarbons (such as benzene, toluene, o-xylene, m-xylene and p-xylene ); chlorinated alkanes (such as chloroform and methylene chloride); dialkyl (such as di-C _1-6 alkyl) ketones (such as acetone, methyl isobutyl ketone), acetonitrile, dimethylformamide, di alkyl ethers (such as diethyl ether, diisopropyl ether, di-n-propyl ether, di-n-butyl ether, and tert-butyl methyl ether); and/or aqueous solvents such as water. Mixtures of any of the above solvents may be used.

Different salts may have different solubilities in any given solvent at any given temperature. In this regard, the compounds of the invention may be readily soluble in some solvents, including some of the solvents described above, and may have less solubility in others. Solvents in which compounds of the present invention are poorly soluble may be referred to as "anti-solvents".

Suitable solvents in which the compounds of the present invention are readily soluble include lower alkyl alcohols such as methanol, ethanol and isopropanol. Lower alkyl acetates (such as ethyl acetate and isopropyl acetate), lower dialkyl ketones (such as methyl isobutyl ketone), aliphatic hydrocarbons (such as isooctane and n-heptane) and aromatic hydrocarbons can be used (such as toluene) as an anti-solvent.

Crystallization of a compound of the invention from a suitable solvent system can be achieved by achieving supersaturation in a solvent system comprising a compound of the invention (eg, by cooling, by evaporation of the solvent and/or by addition of anti-solvent).

The crystalline compounds of the invention (especially crystalline compounds A, B and C) are preferably provided by one or more of the following methods:

(i) preparing an amorphous form of the compound of the present invention and then dissolving the salt in a suitable solvent system such as a polar solvent (e.g. lower alkyl alcohols, lower alkyl acetates, lower dialkyl ketones or mixtures of these solvents) , followed by crystallization (optionally induced by seeding). Crystallization can be carried out by dissolving the compound of the present invention in a solvent in which it is readily soluble (such as a lower alkyl alcohol), and then adding an anti-solvent (such as a lower alkyl acetate or a lower dialkyl ketone), or dissolving the compound in a mixture of a solvent and an anti-solvent in which it is readily soluble, followed by crystallization; or

(ii) Reactive crystallization (or precipitation), comprising adding an appropriate amount of acid to the compound of formula I, then:

(a) direct crystallization, for example from a solvent system comprising an anti-solvent (eg lower alkyl acetate, lower dialkyl ketone or hydrocarbon); or

(b) followed by addition of a suitable anti-solvent to aid in crystallization (e.g. forming the compound of the invention in a solvent in which it is readily soluble (e.g. lower alkyl alcohol) followed by addition of an anti-solvent (e.g. acetate, lower alkyl ketone or hydrocarbon)),

In methods (a) and (b), the acid and/or base can first be provided to a suitable solvent system, and in methods (a) and (b), crystallization can be induced by adding seed crystals.

For method (i) above, preferred solvents may include methyl isobutyl ketone, isopropanol, ethyl acetate, isopropyl acetate, and mixtures thereof.

For method (ii) above, depending on the salt to be formed:

(a) preferred solvents for "direct" crystallization may include isopropanol, isopropyl acetate, n-butyl acetate, toluene or preferably methyl isobutyl ketone or ethyl acetate; and

(b) When an anti-solvent is used for crystallization, the preferred solvent in which the compound of the present invention is easily soluble may include methanol, ethanol or preferably isopropanol; the preferred anti-solvent may include methyl isobutyl ketone, n-butyl acetate, toluene , isooctane, n-heptane or preferably ethyl acetate or isopropyl acetate.

In either method (i) or (ii), those skilled in the art will understand that after salt formation, at least a portion of the solvent may be removed and the resulting mixture redissolved prior to crystallization as described herein.

When the crystalline compound of the invention to be formed is the esylate salt of Compound A, and:

(1) when the method performed is method (i), the salt in amorphous form may be slurried in a mixture of methyl isobutyl ketone or isopropanol and ethyl acetate; and

(2) When the method carried out is method (ii), it can be obtained by adding ethanesulfonic acid (optionally in the form of a solution in methyl isobutyl ketone) to the solution of compound A in methyl isobutyl ketone for direct crystallization. Alternatively, ethanesulfonic acid can be added to a solution of Compound A in isopropanol, and then ethyl acetate can be added as an anti-solvent.

When the crystalline compound of the invention to be formed is the n-propanesulfonate salt of Compound A, and:

(I) when the process performed is process (i), the salt in amorphous form may be slurried in a mixture of isopropanol and isopropyl acetate or a mixture of isopropanol and ethyl acetate; and

(II) When the method carried out is method (ii), n-propanesulfonic acid can be added to the solution of compound A in isopropanol, and then ethyl acetate or isopropyl acetate can be added as anti-solvent.

When the crystalline compound of the invention to be formed is the besylate salt of Compound A, and:

(A) when the method performed is method (i), the salt in the amorphous form may be slurried in ethyl acetate, methyl isobutyl ketone or isopropyl acetate; and

(B) When the method performed is method (ii), benzenesulfonic acid can be added to a solution of compound A in ethyl acetate, followed by the addition of a small amount of isopropanol to facilitate the conversion to the crystalline material. Alternatively, benzenesulfonic acid can be added to a solution of Compound A in isopropanol, followed by addition of ethyl acetate as anti-solvent.

According to another aspect of the invention, the present invention provides a process for the preparation of a crystalline compound of the invention comprising crystallizing a compound of the invention from a suitable solvent system.

The crystallization temperature and crystallization time depend on the salt to be crystallized, the concentration of the salt in solution, and the solvent system used.

Crystallization may also be initiated and/or achieved by standard techniques, eg seeding with or without the addition of crystals of a suitable crystalline compound of the invention.

Compounds of the invention which are anhydrates contain not more than 3%, preferably 2%, more preferably 1% and more preferably 0.5% (w/w) of water, whether such water is bound (water of crystallization, etc.) or unbound.

The different crystalline forms of the compounds of the invention can be conveniently characterized using X-ray powder diffraction (XRPD) methods, for example as described below.

In order to ensure that a particular crystalline form is obtained in the absence of other crystalline forms, crystallization is preferably carried out by adding as seeds nuclei and/or seeds of the desired crystalline form substantially completely free of nuclei and/or seeds of other crystalline forms. Seeds of appropriate compounds may be prepared, for example, by slow evaporation of the solvent from a portion of the appropriate salt solution.

Compounds of the invention may be isolated using techniques well known to those skilled in the art, such as decantation, filtration or centrifugation.

Compounds can be dried using standard techniques.

Additional purifications of compounds of the invention can be performed using techniques well known to those skilled in the art. For example, impurities can be removed by recrystallization from a suitable solvent system. Suitable temperatures and times for recrystallization depend on the concentration of the salt in solution and the solvent system used.

When a compound of the invention is crystallized or recrystallized as described herein, the resulting salt may be in a form having the improved chemical and/or solid state stability described above.

Pharmaceutical formulations and medical applications

Compounds of the invention can be administered to mammalian patients, including humans, and can then be metabolized in vivo to form pharmacologically active compounds (ie, they function as "prodrugs" of the active compound).

Accordingly, the compounds of the present invention are useful because they are metabolized in vivo to form pharmacologically active compounds following oral or parenteral administration. Therefore, the compounds of the present invention are useful as medicines.

Thus, according to another aspect of the invention, the present invention provides a compound of the invention for use as a medicament.

In particular, the compounds of the invention are metabolized to potent thrombin inhibitors after administration, as can be found, for example, in inter alia International Patent Application PCT/SE01/02657 and International Patent Applications WO 02/14270, WO 01/87879 and WO 00/42059 demonstrated in the tests described, said document being incorporated herein by reference.

"Prodrugs of thrombin inhibitors" include compounds that form experimentally detectable amounts of thrombin inhibitors within a predetermined time (eg, about 1 hour) following oral or parenteral administration.

Accordingly, the compounds of the present invention are expected to be useful in conditions where inhibition of thrombin is required and/or where anticoagulant therapy is indicated, including the following:

Treatment and/or prevention of thrombosis and hypercoagulability in the blood and/or tissues of animals, including humans. Hypercoagulability is known to lead to thromboembolic disease. Conditions associated with hypercoagulable and thromboembolic diseases that may be mentioned include hereditary or acquired activator protein C resistance, such as Factor V-mutation (Factor V Leiden), and hereditary or acquired antithrombin, Protein C, protein S, and heparin cofactor II deficiencies. Other conditions known to be associated with hypercoagulable and thromboembolic disorders include circulating antiphospholipid antibodies (lupus anticoagulant), homocysteinemia, heparin-induced thrombocytopenia and insufficient fibrinolysis, and coagulation syndrome symptoms (such as disseminated intravascular coagulation (DIC)) and usually vascular damage (such as due to surgery).

Treatment of conditions in which there is undesired excess thrombin, but no signs of hypercoagulability, such as neurodegenerative diseases such as Alzheimer's disease.

Specific conditions that may be mentioned include the treatment and/or prophylaxis of venous thrombosis (eg DVT) and pulmonary embolism, arterial thrombosis (eg in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis), and systemic embolism, which typically arises from the atrium during atrial fibrillation (eg, nonvalvular atrial fibrillation), or from the left ventricle following a transmural myocardial infarction, or due to congestive heart failure Caused; prevention of reocclusion after thrombolysis, percutaneous transluminal angioplasty (PTA) and coronary artery bypass surgery; prevention of rethrombosis after usual microsurgery and vascular procedures.

Additional indications include treatment and/or prophylaxis of disseminated intravascular coagulation caused by bacteria, polytrauma, poisoning or any other mechanism; Anticoagulant therapy when a body valve or any other medical device is in contact; and anticoagulant therapy when blood is in contact with an extracorporeal medical device, such as during cardiovascular surgery using cardiopulmonary machinery or during hemodialysis; treatment and/or Prevention of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following radiotherapy or chemotherapy, septic shock, sepsis, inflammatory reactions including but not limited to edema, acute or chronic atherosclerosis such as coronary artery disease and atherosclerosis sclerotic plaque formation, cerebral arterial disease, cerebral infarction, cerebral thrombosis, cerebral embolism, peripheral arterial disease, ischemia, angina (including unstable angina), reperfusion injury, percutaneous transluminal angioplasty (PTA) and restenosis after coronary artery bypass surgery.

Compounds of the invention that inhibit trypsin and/or thrombin are also useful in the treatment of pancreatitis.

The compounds according to the invention are therefore indicated for the therapeutic and/or prophylactic treatment of these conditions.

According to another aspect of the invention, the invention provides a method of treating a condition requiring inhibition of thrombin comprising administering to a human suffering from or susceptible to such a condition a therapeutically effective amount of a compound of the invention.

The compounds of the present invention are generally administered orally, intravenously, subcutaneously, buccally, rectally, transdermally, nasally, in the form of pharmaceutical formulations comprising the compounds of the present invention in pharmaceutically acceptable dosage forms Drugs, transtracheal, transbronchial, by other parenteral routes or by inhalation.

The compounds of the present invention may also be co-administered and/or co-administered with antithrombotic agents having a different mechanism of action, such as one or more of the following active agents: antiplatelet agents such as acetylsalicylic acid, thiophene Clopidine and clopidogrel; thromboxane receptor and/or synthetase inhibitors; fibrinogen receptor antagonists; prostacyclin mimetics; phosphodiesterase inhibitors; ADP-receptor (P ₂ T) Antagonists; and Carboxypeptidase U (CPU) inhibitors.

The compounds of the invention may also be administered in combination and/or co-administered with thrombolytic agents, such as one or more tissue plasminogen activators (natural , recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activator, etc.

According to another aspect of the present invention, the present invention provides a pharmaceutical formulation comprising the compound of the present invention in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

In the treatment of humans, suitable daily doses of the compounds of the invention are 0.001-100 mg/kg body weight (in oral administration) and 0.001-50 mg/kg body weight (in parenteral administration), said amounts not including The weight of any acid counterion.

For the avoidance of doubt, the term "treatment" as used herein includes curative and/or prophylactic treatment.

The compounds of the present invention may have the following advantages: they may have higher potency, have lower toxicity, longer duration of action, broader spectrum of activity, be more effective, produce more Fewer side effects, more readily absorbed, and/or have better pharmacokinetic properties (e.g., higher oral bioavailability and/or lower clearance), and/or have other useful pharmacological, physical, or chemical properties . The compounds of the invention may have the additional advantage that they may be administered less frequently than compounds known in the prior art.

The compounds of the invention may also have the advantage that they are provided in a form with improved ease of handling. Furthermore, the compounds of the present invention have the advantage that they can be prepared in a form which may have improved chemical and/or solid state stability, including for example due to lower hygroscopicity. Accordingly, such compounds of the present invention may be stable when stored for extended periods of time.

The compounds of the invention are also advantageous in that they can be crystallized in good yields, in high purity, rapidly, conveniently and at low cost.

The present invention is illustrated by the following examples in conjunction with the accompanying drawings, but in no way limits the present invention, wherein:

Figure 1 shows the X-ray powder diffraction pattern of Compound A ethanesulfonate crystals.

Figure 2 shows the X-ray powder diffraction pattern of compound A besylate salt crystals.

Figure 3 shows the X-ray powder diffraction pattern of compound A n-propanesulfonate crystals.

Figure 4 shows the X-ray powder diffraction pattern of compound A n-butanesulfonate crystals.

Figure 5 shows the X-ray powder diffraction pattern of compound B hemi-1,5-naphthalene disulfonate crystals.

general operation

TLC was performed on silica gel. Chiral HPLC analysis was performed using a 46mm x 250mm Chiralcel OD column with a 5cm guard column. The column temperature was maintained at 35°C. A flow rate of 1.0 mL/min was used. A Gilson 115 UV detector was used at 228nm. For each compound, the mobile phase consisting of hexane, ethanol, and trifluoroacetic acid is listed along with the appropriate ratios. In general, the product is dissolved in a minimal amount of ethanol and diluted with the mobile phase.

In Prep. AC below, LC-MS/MS was performed using an HP-1100 apparatus equipped with a CTC-PAL injector and a 5Tm, 4 x 100mm ThermoQuest, Hypersil BDS-C18 column. An API-3000 (Sciex) MS detector was used. The flow rate was 1.2 mL/min and the mobile phase (gradient) consisted of 10-90% acetonitrile and 90-10% 4 mM ammonium acetate in water, both containing 0.2% formic acid. In addition, low-resolution mass spectra (LRMS) were recorded using a Micromass ZQ spectrometer with ESI positive and negative ionization (mass range m/z 100-800); high-resolution mass spectra (HRMS) were recorded with ES negative ionization using a Micromass LCT spectrometer Recorded (mass range m/z 100-1000), using leucine enkephalin (C ₂₈ H ₃₇ N ₅ O ₇ ) as mass internal standard.

¹ H NMR spectra were performed using tetramethylsilane as an internal standard. ¹³ C NMR spectra were performed using the listed deuterated solvents as internal standards. Otherwise, MeOD was used as solvent and the MeOD signal was used as internal standard ( ¹ HΛ = 3.30 ppm; ¹³ CΛ = 49 ppm).

X-ray powder diffraction analysis (XRPD) was carried out using variable slits on samples prepared according to standard methods, with and without any internal standard, such as those described in: Giacovazzo, C. et al. (1995), Fundamentals of Crystallography, Oxford University Press; Jenkins, R. and Snyder, R.L. (1996), Introduction to X-Ray Powder Diffractometry, John Wiley & Sons, New York; Bunn, C.W. (1948), Chemical Crystallography, Clarendon Press, London; or Klug, H.P. & Alexander, L.E. (1974), X-ray Diffraction Procedures, John Wiley and Sons, New York. X-ray analysis was performed with a Siemens D5000 diffractometer and a Philips X'Pert MPD.

Differential Scanning Calorimetry (DSC) was performed with a Mettler DSC820 apparatus according to standard methods such as those described in: Hohne, G.W.H. et al. (1996), Differential Scanning Calorimetry, Springer, Berlin.

Thermogravimetric analysis (TGA) was performed with a Mettler Toledo TGA850 apparatus.

Those skilled in the art should understand that the crystalline form of the compound of the present invention can be prepared by methods similar to those described herein and/or according to the examples below, and can exhibit substantially the same XRPD diffraction pattern and/or as disclosed herein Or DSC and/or TGA differential thermogram. "Substantially identical" XRPD diffraction patterns and/or DSC and/or TGA thermograms include, when clearly seen from the associated pattern and/or thermogram (allowing for experimental error), substantially Examples of the same crystal form above. As supplied, DSC onset temperatures may vary within ±5°C (eg, ±2°C), and XRPD distance values may vary within ±2 to the last decimal place. Those skilled in the art will appreciate that when determining substantially identical crystalline forms, XRPD intensities can vary for a variety of reasons including, for example, preferred orientation.

XRPD data intensities are typically within a margin of error of about ±20-40%. Relative intensities can be characterized according to the following definitions:

% Relative Strength Definition

60-100 vs (very strong)

21-59.9 s (strong)

7-20.9 m (medium)

4-6.9 w (weak)

<1-3.9 vw (very weak)

In the Examples section, unless otherwise stated, when seeds were added, the seeds were obtained from the first Example in which crystals of the salt were obtained. For example, in Example 13, the seed crystals were obtained from Example 11.

Preparation A: Preparation of Compound A

(i) 3-Chloro-5-methoxybenzaldehyde

3,5-Dichloroanisole (74.0 g, 419 mmol) in THF (200 mL) was added dropwise to magnesium metal (14.2 g, 585 mmol) in THF (100 mL), prewashed with 0.5N HCl at 25 °C over). After the addition, 1,2-dibromoethane (3.9 g, 20.8 mmol) was added dropwise. The resulting dark brown mixture was heated to reflux for 3 hours. The mixture was cooled to 0°C and N,N-dimethylformamide (60 mL) was added in one portion. The mixture was partitioned with ether (3 x 400 mL) and 6N HCl (500 mL). The combined organic extracts were washed with brine (300 mL), dried ( _Na2SO4 ), filtered and concentrated in _vacuo to an oil. Purification by flash chromatography on silica gel (2x) eluting with Hex:EtOAc (4:1) afforded the subtitle compound (38.9 g, 54%) as a yellow oil.

¹ H NMR (300MHz, CDCl ₃ ) δ9.90(s, 1H), 7.53(s, 1H), 7.38(s, 1H), 7.15(s, 1H), 3.87(s, 3H),

(ii) 3-Chloro-5-hydroxybenzaldehyde

A solution of 3-chloro-5-methoxybenzaldehyde (22.8 g, 134 mmol; see step (i) above) in _CH2Cl2 (250 _mL ) was cooled to 0 °C. Boron tribromide (15.8 mL, 167 mmol) was added dropwise over 15 minutes. After stirring the reaction mixture for 2 h, _H2O (50 mL) was added slowly. The solution was then extracted with _Et2O (2 x 100 mL). _The organic layers were combined, dried ( _Na2SO4 ), filtered and concentrated in vacuo. Purification by flash chromatography on silica gel eluting with Hex:EtOAc (4:1) afforded the subtitle compound (5.2 g, 25%).

¹ H NMR (300MHz, CDCl ₃ ) δ9.85(s, 1H), 7.35(s, 1H), 7.20(s, 1H), 7.10(s, 1H), 3.68(s, 1H)

(iii) 3-fluoro-5-difluoromethoxybenzaldehyde

A solution of 3-chloro-5-hydroxybenzaldehyde (7.5 g, 48 mmol; see step (ii) above) in 2-propanol (250 mL) and 30% KOH (100 mL) was heated to reflux. With stirring, _CHClF2 was bubbled through the reaction mixture for 2 hours. The reaction mixture was cooled, acidified with 1N HCl, and extracted with EtOAc (2 x 100 mL). The organic layer was washed with brine (100 mL), dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. Purification by flash chromatography on silica gel eluting with Hex:EtOAc (4:1) afforded the subtitle compound (4.6 g, 46%).

¹ H NMR (300MHz, CDCl ₃ ) δ9.95(s, 1H), 7.72(s, 1H), 7.52(s, 1H), 7.40(s, 1H), 6.60(t, J _HP = 71.1Hz, 1H )

(iv) Ph(3-Cl)(5-OCHF ₂ )-(R,S)CH(OTMS)CN

A solution of 3-chloro-5-difluoromethoxybenzaldehyde (4.6 g, 22.3 mmol; see step (iii) above) in _CH2Cl2 (200 _mL ) was cooled to 0 °C. _ZnI2 (1.8 g, 5.6 mmol) and trimethylsilyl cyanide (2.8 g, 27.9 mmol) were added and the reaction mixture was allowed to warm to room temperature and stirred for 15 hours. The mixture was partially concentrated under vacuum to afford the subtitle compound as a liquid which was used in step (v) below without further purification or characterization.

(v) Ph(3-Cl)(5-OCHF ₂ )-(R,S)CH(OH)C(NH)OEt

Ph(3-Cl)(5- _OCHF2 )-(R,S)CH(OTMS)CN (6.82 g, assumed 22.3 mmol; see step (iv) above) was added dropwise to HCl/EtOH (500 mL) . The reaction mixture was stirred for 15 hours and then partially concentrated under vacuum to afford the subtitle compound as a liquid which was used directly in step (vi) below without further purification or characterization.

(vi) Ph(3-Cl)(5-OCHF ₂ )-(R,S)CH(OH)C(O)OEt

Ph(3-Cl)(5- _OCHF2 )-(R,S)CH(OH)C(NH)OEt (6.24 g, assume 22.3 mmol; see step (v) above) was dissolved in THF (250 mL) , 0.5M _H2SO4 (400 _mL ) was added and the reaction was stirred at 40 °C for 65 hours, cooled, then partially concentrated in vacuo to remove most of the THF. The reaction mixture was then extracted with _Et2O (3 x 100 mL), dried ( _Na2SO4 ), filtered and concentrated _in vacuo to afford the subtitle compound as a solid which was used without further purification or characterization in the following In step (vii).

(vii) Ph(3-Cl)(5-OCHF ₂ )-(R,S)CH(OH)C(O)OH

Ph(3-Cl)(5- _OCHF2 )-(R,S)CH(OH)C(O)OEt (6.25 g, assumed 22.3 mmol; see step (vi) above) was dissolved in 2-propanol ( 175 mL) and 20% KOH (350 mL) was stirred at room temperature for 15 hours. Partially concentrated under vacuum. The reaction was then partially concentrated under vacuum to remove most of the 2-propanol. The remaining mixture was _acidified with 1M _H2SO4 , extracted with _Et2O (3 x 100 mL), dried ( _Na2SO4 ) and concentrated in vacuo to give a solid _. Purification by flash chromatography on silica gel eluting with _CHCl3 :MeOH:conc. _NH4OH (6:3:1) afforded the ammonium salt of the subtitle compound. The ammonium salt was then dissolved in a mixture of EtOAc (75 mL) and _H2O (75 mL) and acidified with 2N HCl. The organic layer was separated, washed with brine (50 mL), dried ( _Na2SO4 ) and concentrated in vacuo to afford the subtitle compound ( _3.2 g, 57% yield from steps (iv) to (vii)).

¹ H NMR (300MHz, CD ₃ OD) δ7.38(s, 1H), 7.22(s, 1H), 7.15(s, 1H), 6.89(t, _JH-F =71.1Hz, 1H), 5.16( s, 1H)

(viii) Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)OH(a) and Ph(3-Cl)(5-OCHF ₂ )-(S)CH( OAc)C(O)OH(b)

Ph(3-Cl)(5- _OCHF2 )-(R,S)CH(OH)C(O)OH (3.2 g, 12.7 mmol; see step (vii) above) and Lipase PS "Amano" ( ~2.0 g) in vinyl acetate (125 mL) and MTBE (125 mL) was heated at reflux for 48 hours. The reaction mixture was cooled, filtered through celite, and the filter cake was washed with EtOAc. The filtrate was concentrated in vacuo and purified by flash silica gel chromatography eluting with _CHCl3 :MeOH:conc. _NH4OH (6:3:1) to afford the ammonium salts of the subtitled compounds (a) and (b). The salt of compound (a) was dissolved in _H2O , acidified with 2N HCl, extracted with EtOAc. The organic layer was washed with brine, dried ( _Na2SO4 ), filtered and concentrated _in vacuo to afford the subtitled compound (a) (1.2 g, 37%).

For this subtitle compound (a)

¹ H NMR (300MHz, CD ₃ OD) δ7.38(8, 1H), 7.22(s, 1H), 7.15(s, 1H), 6.89(t, JH _-F =71.1Hz, 1H), 5.17( s, 1H)

(ix)Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-Aze-Pab(Teoc)

Add Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)OH (1.1 g, 4.4 mmol; see step (viii) above) and H-Aze- To a solution of Pab(Teoc) (see International Patent Application WO 00/42059, 2.6 g, 5.7 mmol) in DMF (50 mL) was added PyBOP (2.8 g, 5.3 mmol) and collidine (1.3 g, 10.6 mmol). The reaction was stirred at 0 °C for 2 hours, then at room temperature for an additional 15 hours. The reaction mixture was concentrated in vacuo and purified by flash silica gel chromatography (3x), eluting first with _CHCl3 :EtOH (9: ₁ ), then EtOAc:EtOH (20:1) and finally _CH2Cl2 Elution with : _CH3OH (95:5) afforded the subtitle compound (1.0 g, 37%) as a white amorphous solid.

¹ H NMR (300 MHz, CD ₃ OD, mixture of rotamers) δ 7.79-7.85 (d, J = 8.7 Hz2H), 7.15-7.48 (m, 5H), 6.89 and 6.91 (t, J _HF = 71.1 Hz , IH), 5.12 and 5.20 (s, 1H), 4.75-4.85 (m, 1H), 3.97-4.55 (m, 6H), 2.10-2.75 (m, 2H), 1.05-1.15 (m, 2H), 0.09 (s, 9H)

MS(m/z)611(M+1) ⁺

(x)Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-Aze-Pab(OMe, Teoc)

Dissolve Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)-Aze-Pab(Teoc) (0.40 g, 0.65 mmol; see step (ix) above) in 20 mL In acetonitrile, 0.50 g (6.0 mmol) of O-methylhydroxylamine hydrochloride was added. The mixture was heated at 70°C for 2 hours. The solvent was evaporated, the residue was partitioned between water and ethyl acetate, the aqueous phase was extracted twice with ethyl acetate, the combined organic phases were washed with water, brine, dried ( _Na2SO4 ), filtered and evaporated _. Yield: 0.41 g (91%).

¹ H-NMR. (400MHz; CDCl ₃ ): δ7.83 (bt, 1H), 7.57 (bs, 1H), 7.47 (d, 2H), 7.30 (d, 2H), 7.20 (m, 1H), 7.14 (m, 1H), 7.01(m, 1H), 6.53(t, 1H), 4.89(s, 1H), 4.87(m, 1H), 4.47(m, 2H), 4.4-4.2(b, 1H), 4.17-4.1(m, 3H), 3.95(s, 3H), 3.67(m, 1H), 2.68(m, 1H), 2.42(m, 1H), 0.97(m, 2H), 0.01(s, 9H),

(xi) Compound A

Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)-Aze-Pab(OMe, Teoc) (0.40 g, 0.62 mmol; see step (x) above) was dissolved In 5 mL of TFA, let the reaction proceed for 30 min. TFA was evaporated and the residue was partitioned between ethyl acetate and _NaHCO3 (aq). The aqueous phase was extracted twice with ethyl acetate and the combined organic phases were washed with water, brine, dried ( _Na2SO4 ), filtered and _evaporated . The product was lyophilized from water/acetonitrile. No purification was required. Yield: 0.28 g (85%).

¹ H-NMR (600MHz; CDCl ₃ ): δ7.89 (bt, 1H), 7.57 (d, 2H), 7.28 (d, 2H), 7.18 (m, 1H), 7.13 (m, 1H), 6.99 ( m, 1H), 6.51(t, 1H), 4.88(s, 1H), 4.87(m, 1H), 4.80(bs, 2H), 4.48(dd, 1H), 4.43(dd, 1H), 4.10(m , 1H), 3.89(s, 3H), 3.68(m, 1H), 2.68(m, 1H), 2.40(m, 1H).

¹³ C-NMR (125MHz; CDCl ₃ ): (carbonyl and/or amidine carbon, rotamer) δ172.9, 170.8, 152.7, 152.6

_HRMS , Calcd. _{for C22H23ClF2N4O5} (MH) ^- _495.1242 , found _495.1247

Preparation B: Preparation of Compound B

(i) 2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile

Under argon atmosphere, (methylsulfinyl)(methylthio)methane (7.26 g, 0.0584 mol) was dissolved in 100 mL of anhydrous THF and cooled to -78°C. A solution of butyllithium in hexane (16 mL of 1.6M, 0.0256 mol) was added dropwise with stirring. The mixture was stirred for 15 minutes. Meanwhile, under an argon atmosphere, a solution of 3,4,5-trifluorobenzonitrile (4.0 g, 0.025 mmol) in 100 mL of anhydrous THF was cooled to -78 °C, and the former solution was added to in the latter solution. After 30 minutes, the cooling bath was removed and when the reaction reached room temperature, it was poured into 400 mL of water. THF was evaporated and the remaining aqueous layer was extracted 3 times with ether. The combined ether phases were washed with _water , dried ( _Na2SO4 ) and evaporated. Yield: 2.0 g (30%).

¹ H NMR (500MHz, CDCl ₃ ) δ7.4-7.25 (m, 2H), 5.01 (s, 1H, diastereomer), 4.91 (s, 1H, diastereomer), 2.88 (s, 3H, diastereomer), 2.52 (s, 3H, diastereomer), 2.49 (s, 3H, diastereomer), 2.34 (s, 3H, diastereomer), 1.72 (broad peak, 1H)

(ii) 2,6-difluoro-4-formylbenzonitrile

2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile (2.17 g, 8.32 mmol; see step (i) above) was dissolved in 90 mL THF and 3.5 mL concentrated sulfuric acid. The mixture was left at room temperature for 3 days, then poured into 450 mL of water. Extracted 3 times with EtOAc, the combined ethyl acetate phases were washed 2 times with aqueous sodium bicarbonate solution, washed with brine, _dried ( _Na2SO4 ) and evaporated. Yield: 1.36 g (98%). The position of the formyl group was determined ^by13C NMR. The signal of the fluorocarbon at 162.7 ppm exhibits the expected coupling pattern, with two coupling constants of the order 260 Hz and 6.3 Hz, corresponding to the native and meta coupling from the fluorine atom, respectively.

¹ H NMR (400MHz, CDCl ₃ ) δ10.35(s, 1H), 7.33(m, 2H)

(iii) 2,6-difluoro-4-hydroxymethylbenzonitrile

2,6-Difluoro-4-formylbenzonitrile (1.36 g, 8.13 mmol; see step (ii) above) was dissolved in 25 mL of methanol and cooled on an ice bath. Sodium borohydride (0.307 g, 8.12 mmol) was added portionwise with stirring and the reaction was left for 65 minutes. The solvent was evaporated and the residue was partitioned between diethyl ether and aqueous sodium bicarbonate. The ether layer was washed with _aqueous sodium bicarbonate and brine, dried ( _Na2SO4 ) and evaporated. The crude product crystallized quickly and was used without further purification. Yield: 1.24 g (90%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.24(m, 2H), 4.81(s, 2H), 2.10 (broad peak, 1H)

(iv) 4-cyano-2,6-difluorobenzyl methanesulfonate

Add 2,6-difluoro-4-hydroxymethylbenzonitrile (1.24 g, 7.32 mmol; see step (iii) above) and methanesulfonyl chloride (0.93 g, 8.1 mmol) in 60 mL of dichloromethane under stirring Triethylamine (0.81 g, 8.1 mmol) was added to the ice-cold solution in . After 3 h at 0°C, the mixture was washed twice with 1M HCl, once with water, dried ( _Na2SO4 ) and evaporated _. The product was used without further purification. Yield: 1.61 g (89%).

¹ H NMR (300MHz, CDCl ₃ ) δ7.29(m, 2H), 5.33(s, 2H), 3.07(s, 3H)

(v) 4-azidomethyl-2,6-difluorobenzonitrile

4-cyano-2,6-difluorobenzyl methanesulfonate (1.61 g, 6.51 mmol; see step (iv) above) and sodium azide (0.72 g, 0.0111 mol) in 10 mL of water and 20 mL The mixture in DMF was stirred overnight at room temperature. The resulting mixture was then poured into 200 mL of water and extracted 3 times with ether. The combined ether phases were washed 5 times with _water , dried ( _Na2SO4 ) and evaporated. A small sample was evaporated for NMR determination and the product crystallized. The remaining product was carefully evaporated, but not completely to dryness. The yield (theoretical yield 1.26 g) was assumed to be almost quantitative based on NMR and analytical HPLC.

¹ H NMR (400MHz, CDCl ₃ ) δ7.29(m, 2H), 4.46(s, 2H)

(vi) 4-Aminomethyl-2,6-difluorobenzonitrile

The reaction was carried out according to the method described in J. Chem. Res. (M) (1992) 3128 . To a suspension of 520 mg of 10% Pd/C (50% moisture) in 20 mL of water was added a solution of sodium borohydride (0.834 g, 0.0221 mol) in 20 mL of water. As a result certain gases are released. 4-Azidomethyl-2,6-difluorobenzonitrile (1.26 g, 6.49 mmol; see step (v) above) was dissolved in 50 mL THF and added to the aqueous mixture on an ice bath over 15 minutes middle. The mixture was stirred for 4 hours, then 20 mL of 2M HCl was added and the mixture was filtered through celite. The celite was washed again with water and the combined aqueous phases were washed with EtOAc and then basified with 2M NaOH. Extracted 3 times with dichloromethane, the combined organic phases were washed with water, _dried ( _Na2SO4 ) and evaporated. Yield: 0.87 g (80%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.20(m, 2H), 3.96(s, 2H), 1.51 (broad peak, 2H)

(vii) 2,6-difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile

4-Aminomethyl-2,6-difluorobenzonitrile (0.876 g, 5.21 mmol; see step (vi) above) was dissolved in 50 mL THF and di-tert-butyl dicarbonate (1.14 g, 5.22 mmol). The mixture was stirred for 3.5 hours. THF was evaporated and the residue was partitioned between water and EtOAc. The organic layer was _washed 3 times with 0.5M HCl and water, dried ( _Na2SO4 ) and evaporated. The product was used directly without further purification. Yield: 1.38 g (99%).

¹ H NMR (300MHz, CDCl ₃ ) δ7.21(m, 2H), 4.95(broad peak, 1H), 4.43(broad peak, 2H), 1.52(s, 9H)

(viii) Boc-Pab(2,6-diF)(OH)

2,6-Difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile (1.38 g, 5.16 mmol; see step (vii) above), hydroxylamine hydrochloride (1.08 g, 0.0155 mol) and tris A mixture of ethylamine (1.57 g, 0.0155 mol) in 20 mL of ethanol was stirred at room temperature for 36 hours. The solvent was evaporated and the residue was partitioned between water and dichloromethane. The organic layer was washed with _water , dried ( _Na2SO4 ) and evaporated. The product was used directly without further purification. Yield: 1.43 g (92%).

¹ H NMR (500MHz, CD ₃ OD) δ7.14(m, 2H), 4.97(broad peak, 1H), 4.84(broad peak, 2H), 4.40(broad peak, 2H), 1.43(s, 9H)

(ix) Boc-Pab(2,6-diF)×HOAc

The reaction was carried out according to the method described by Judkins et al., Synth. Comm. (1998) 4351 . Boc-Pab(2,6-diF)(OH) (1.32 g, 4.37 mmol; see step (viii) above), acetic anhydride (0.477 g, 4.68 mmol) and 442 mg of 10% Pd in 100 mL of acetic acid /C (50% moisture) hydrogenation at 5 atm pressure for 3.5 hours. The mixture was filtered through celite, washed with ethanol, and evaporated. The residue was lyophilized from acetonitrile and water with a few drops of ethanol and the subtitle product was used without further purification. Yield: 1.49 g (99%).

¹ H NMR (400MHz, CD ₃ OD) δ7.45(m, 2H), 4.34(s, 2H), 1.90(s, 3H), 1.40(s, 9H)

(x)Boc-Pab(2,6-diF)(Teoc)

To a solution of Boc-Pab(2,6-diF)×HOAc (1.56 g, 5.49 mmol; see step (ix) above) in 100 mL THF and 1 mL water was added 2-(trimethylsilylcarbonate ) ethyl ester p-nitrophenyl ester (1.67 g, 5.89 mmol). A solution of potassium carbonate (1.57 g, 0.0114 mol) in 20 mL of water was added dropwise over 5 minutes. The mixture was stirred overnight. THF was evaporated and the residue was partitioned between water and dichloromethane. The aqueous layer was extracted with dichloromethane, the combined organic phases were washed twice with aqueous sodium bicarbonate, dried ( _Na2SO4 ) and evaporated _. Purification by flash chromatography on silica gel eluting with heptane/EtOAc=2/1 afforded 1.71 g (73%) of pure compound.

¹ H NMR (400MHz, CDCl ₃ ) δ7.43 (m, 2H), 4.97 (broad peak, 1H), 4.41 (broad peak, 2H), 4.24 (m, 2H), 1.41 (s, 9H), 1.11 ( m, 2H), 0.06 (s, 9H)

(xi) Boc-Aze-Pab(2,6-diF)(Teoc)

Boc-Pab(2,6-diF)(Teoc) (1.009 g, 2.35 mmol; see step (x) above) was dissolved in 50 mL of EtOAc saturated with HCl(g). The mixture was left for 10 minutes, evaporated and dissolved in 18 mL of DMF, then cooled on an ice bath. Boc-Aze-OH (0.450 g, 2.24 mmol), PyBOP (1.24 g, 2.35 mmol) and diisopropylethylamine (1.158 g, 8.96 mmol) were added sequentially. The reaction mixture was stirred for 2 hours, then poured into 350 mL of water and extracted 3 times with EtOAc. The combined organic phases were washed with brine, _dried ( _Na2SO4 ) and evaporated. Purification by flash chromatography on silica gel with heptane:EtOAc (1:3) afforded 1.097 g (96%) of the desired compound.

¹ H NMR (500MHz, CDCl ₃ ) δ7.46(m, 2H), 4.65-4.5(m, 3H), 4.23(m, 2H), 3.87(m, 1H), 3.74(m, 1H), 2.45- 2.3(m, 2H), 1.40(s, 9H), 1.10(m, 2H), 0.05(s, 9H)

(xii) Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc)

Boc-Aze-Pab(2,6-diF)(Teoc) (0.256 g, 0.500 mmol; see step (xi) above) was dissolved in 20 mL of EtOAc saturated with HCl(g). The mixture was left for 10 minutes, evaporated and dissolved in 5 mL DMF. Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)OH (0.120 g, 0.475 mmol; see Preparation A(viii)), PyBOP (0.263 g, 0.498 mmol) were added sequentially and diisopropylethylamine (0.245g, 1.89mmol). The reaction mixture was stirred for 2 hours, then poured into 350 mL of water and extracted 3 times with EtOAc. The combined organic phases were washed with brine, _dried ( _Na2SO4 ) and evaporated. Purification by flash silica gel chromatography eluting with EtOAc afforded 0.184 g (60%) of the desired subtitle compound.

¹ H NMR (400MHz, CD ₃ OD, mixture of rotamers) δ 7.55-7.45(m, 2H), 7.32(m, 1H, major rotamer), 7.27(m, 1H, minor rotamer isomer), 7.2-7.1(m, 2H), 6.90(t, 1H, major rotamer), 6.86(t, 1H, minor rotamer), 5.15(s, 1H, major rotamer isomer), 5.12 (m, 1H, minor rotamer), 5.06 (s, 1H, minor rotamer), 4.72 (m, 1H, major rotamer), 4.6-4.45 (m, 2H), 4.30 (m, 1H, major rotamer), 4.24 (m, 2H), 4.13 (m, 1H, major rotamer), 4.04 (m, 1H, minor rotamer), 3.95(m, 1H, minor rotamer), 2.62(m, 1H, minor rotamer), 2.48(m, 1H, major rotamer), 2.22(m, 1H, major rotamer isomer), 2.10 (m, 1H, minor rotamer), 1.07 (m, 2H), 0.07 (m, 9H)

(xiii) Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(OMe, Teoc)

Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(Teoc) (64 mg, 0.099 mmol; see procedure above A mixture of (xii)) and O-methylhydroxylamine hydrochloride (50 mg, 0.60 mmol) in 4 mL of acetonitrile was heated at 70° C. for 3 hours. The solvent was evaporated and the residue was partitioned between water and EtOAc. The aqueous layer was extracted twice with EtOAc, the combined organic phases were washed with water, _dried ( _Na2SO4 ) and evaporated. The product was used directly without further purification. Yield: 58 mg (87%).

¹ H NMR (400MHz, CDCl ₃ ) δ7.90(bt, 1H), 7.46(m, 1H), 7.25-6.95(m, 5H), 6.51, t, 1H), 4.88(s, 1H), 4.83( m, 1H), 4.6-4.5(m, 2H), 4.4-3.9(m, 4H), 3.95(s, 3H), 3.63(m, 1H), 2.67(m, 1H), 2.38(m, 1H) , 1.87 (broad peak, 1H), 0.98 (m, 2H), 0.01, s, 9H)

(xiv) Compound B

Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)-Aze-Pab(2,6-diF)(OMe, Teoc) (58 mg, 0.086 mmol; see above Step (xiii)) was dissolved in 3 mL of TFA, cooled on an ice bath, and allowed to react for 2 hours. TFA was evaporated and the residue was dissolved in EtOAc. The organic layer was washed _twice with aqueous sodium carbonate and water, dried ( _Na2SO4 ) and evaporated. The residue was lyophilized from water and acetonitrile to afford 42 mg (92%) of the title compound.

¹ H NMR (300MHz, CDCl ₃ ) δ7.95(bt, 1H), 7.2-7.1(m, 4H), 6.99(m, 1H), 6.52(t, 1H), 4.88(s, 1H), 4.85- 4.75(m, 3H), 4.6-4.45(m, 2H), 4.29(broad peak, 1H), 4.09(m, 1H), 3.89(s, 3H), 3.69(m, 1H), 2.64(m, 1H ), 2.38 (m, 1H), 1.85 (broad peak, 1H) ¹³ C-NMR (100MHz; CDCl ₃ ): (carbonyl and/or amidine carbon) δ172.1, 169.8, 151.9APCI-MS: (M+1 ) = 533/535 m/z

Preparation C: Preparation of Compound C

(i) (2-monofluoroethyl) methanesulfonate

To a magnetically stirred solution of 2-fluoroethanol (5.0 g, 78.0 mmol) in _CH2Cl2 (90 mL) was added triethylamine (23.7 g, ₂₃₄ mmol) and methanesulfonyl chloride at 0 °C under nitrogen atmosphere (10.7 g, 93.7 mmol). The mixture was stirred at 0 °C for 1.5 h _, diluted with _CH2Cl2 (100 mL), washed with 2N HCl (100 mL). The aqueous layer was extracted with _CH2Cl2 (50 mL), the combined organic extracts were washed with brine (75 mL), dried ( _Na2SO4 ), filtered and concentrated _in vacuo to afford the subtitle compound (9.7 _g , 88% ), as a yellow oil which was used without further purification.

¹ H NMR (300MHz, CDCl ₃ ) δ4.76(t, J=4Hz, 1H), 4.64(t, J=4Hz, 1H), 4.52(t, J=4Hz, 1H), 4.43(t, J=4Hz, 1H) 4Hz, 1H), 3.09(s, 3H).

(ii) 3-Chloro-5-monofluoroethoxybenzaldehyde

To a solution of 3-chloro-5-hydroxybenzaldehyde (8.2 g, 52.5 mmol; see Preparation A(ii)) and potassium carbonate (9.4 g, 68.2 mmol) in DMF (10 mL) under nitrogen atmosphere at room temperature A solution of (2-monofluoroethyl)methanesulfonate (9.7 g, 68.2 mmol; see step (i) above) in DMF (120 mL) was added dropwise. The mixture was heated at 100°C for 5 hours, then stirred at room temperature overnight. The reaction was cooled to 0 °C, poured into ice-cold 2N HCl, and extracted with EtOAc. The combined organic extracts _were washed with brine, dried ( _Na2SO4 ), filtered and concentrated in vacuo. Purification of the brown oil by silica gel chromatography eluting with Hex:EtOAc (4:1) afforded the subtitle compound (7.6 g, 71%) as a yellow oil.

¹ H NMR (300MHz, CDCl ₃ ) δ9.92(s, 1H), 7.48(s, 1H), 7.32(s, 1H), 7.21(s, 1H), 4.87(t, J=4Hz, 1H), 4.71(t, J=3Hz, 1H), 4.33(t, J=3Hz, 1H), 4.24(t, J=3Hz, 1H).

(iii) Ph(3-Cl)(5-OCH ₂ CH ₂ F)-(R,S)CH(OTMS)CN

3-Chloro-5-monofluoroethoxybenzaldehyde (7.6 g, 37.5 mmol; see step (ii) above) and zinc iodide (3.0 g, 9.38 mmol) were added under nitrogen atmosphere at 0 °C in CH To a solution in ₂ Cl ₂ (310 mL) was added trimethylsilyl cyanide (7.4 g, 75.0 mmol) dropwise. The mixture was stirred at 0°C for 3 hours and at room temperature overnight. The reaction was diluted with _H2O (300 mL), the organic layer was separated, dried ( _Na2SO4 ), filtered and concentrated _in vacuo to afford the subtitle compound (10.6 g, 94%) as a brown oil which was Used without further purification or characterization.

(iv) Ph(3-Cl)(5- _OCH2CH2F ) _- (R,S)CH(OH)C(O)OH

Concentrated hydrochloric acid (100 mL) was added to Ph(3-Cl)(5- _OCH2CH2F )-(R,S)CH(OTMS)CN (10.6 g, 5.8 mmol; see _step (iii) above) , and the solution was stirred at 100°C for 3 hours. After cooling to room temperature, the reaction was further cooled to 0 °C, slowly basified with 3N NaOH (-300 mL), washed with _Et2O (3 x 200 mL). The aqueous layer was acidified with 2N HCl (80 mL), extracted with EtOAc (3 x 300 mL). The combined EtOAc extracts were dried ( _Na2SO4 ), filtered and concentrated _in vacuo to afford the subtitle compound (8.6 g, 98%) as a pale yellow solid which was used without further purification.

R _f =0.28 (90:8:2 CHCl ₃ :MeOH:conc. NH ₄ OH)

¹ H NMR (300MHz, CD ₃ OD) δ7.09(s, 1H), 7.02(s, 1H), 6.93(s, 1H), 5.11(s, 1H), 4.77-4.81(m, 1H), 4.62 -4.65(m, 1H), 4.25-4.28(m, 1H), 4.15-4.18(m, 1H),

(v) Ph(3-Cl)(5 _{- OCH2CH2F} )-(S)CH(OAc)C(O)OH(a) and Ph(3-Cl)(5 _{- OCH2CH2F} ) -(R)CH(OH)C(O)OH(b)

Ph(3-Cl)(5- _OCH2CH2F )-(R,S)CH(OH)C(O)OH (8.6 g, 34.5 mmol; see step (iv) above) _and Lipase PS" A solution of Amano" (4.0 g) in vinyl acetate (250 mL) and MTBE (250 mL) was heated at 70 °C under nitrogen atmosphere for 3 days. The reaction was cooled to room temperature and the enzyme was removed by filtration through celite. The filter cake was washed with EtOAc and the filtrate was concentrated in vacuo. Purification by silica gel chromatography, eluting with _CHCl3 :MeOH: _Et3N (90:8:2), afforded the triethylamine salt of subtitle compound (a) as a yellow oil. In addition, triethylamine salt (4.0 g) of the subtitle compound (b) was also obtained. The salt of subtitle compound (b) was dissolved in _H2O (250 mL), acidified with 2N HCl, extracted with EtOAc (3 x 200 mL). The combined organic extracts were dried ( _Na2SO4 ), filtered and concentrated _in vacuo to afford the subtitle compound (b) (2.8 g, 32%) as a yellow oil.

Data on this subtitle compound (b):

R _f =0.28 (90:8:2 CHCl ₃ :MeOH:conc. NH ₄ OH)

¹ H NMR (300MHz, CD ₃ OD) δ7.09(s, 1H), 7.02(s, 1H), 6.93(s, 1H), 5.11(s, 1H), 4.77-4.81(m, 1H), 4.62 -4.65(m, 1H), 4.25-4.28(m, 1H), 4.15-4.18(m, 1H).

(vi) Compound C

To Ph(3-Cl)(5- _OCH2CH2F )-(R)CH(OH)C(O)OH (818 mg, 3.29 mmol; see _step (v) above) at 0 °C under nitrogen atmosphere ) to a solution in DMF (30 mL) was added HAze-Pab(OMe).2HCl (1.43 g, 4.27 mmol, see International Patent Application WO00/42059), PyBOP (1.89 g, 3.68 mmol) and DIPEA (1.06 g, 8.23 mmol). The reaction was stirred at 0 °C for 2 hours, then at room temperature overnight. The mixture was concentrated in vacuo and the residue was chromatographed twice on silica gel, eluting first with _CHCl3 :EtOH (15:1) and then with EtOAc:EtOH (20:1) to afford the title compound (880 mg , 54%).

R _f =0.60 (10:1 CHCl ₃ :EtOH)

¹ H NMR (300MHz, CD ₃ OD, complex mixture of rotamers) δ7.58-7.60 (d, J=8Hz, 2H), 7.34 (d, J=7Hz, 2H), 7.05-7.08 (m, 2H), 6.95-6.99(m, 1H), 5.08-5.13(m, 1H), 4.77-4.82(m, 1H), 4.60-4.68(m, 1H), 3.99-4.51(m, 7H), 3.82( s, 3H), 2.10-2.75 (m, 2H).

¹³ C-NMR (150MHz; CD ₃ OD): (carbonyl and/or amidine carbon) δ173.3, 170.8, 152.5. APCI-MS: (M+1) = 493m/z.

Examples 1 and 2: Preparation of Salt of Compound A

Example 1: General method for preparing salts

The following general procedure was used to prepare the salt of Compound A: 200 mg of Compound A (see Preparation A above) was dissolved in 5 mL of MeOH. To this solution was added a solution of the corresponding acid (1.0 molar equivalent) dissolved in 5 mL of MeOH. After stirring at room temperature for 10 minutes, the solvent was removed by rotary evaporator. The remaining solid material was redissolved in 8 mL of acetonitrile: _H2O (1:1). Freeze-drying gave a colorless amorphous material in each case.

Acid used:

(1S)-(+)-10-camphorsulfonic acid

malic acid

cyclamic acid

phosphoric acid

Dimethyl Phosphate

p-Toluenesulfonic acid

L-Lysine

L-Lysine Hydrochloride

Saccharinic acid

Methanesulfonic acid

hydrochloric acid

Suitable characteristic data are shown in Table 1.

Table 1 Salt Mw acid Mw salt LRMS Appm(MeOD)H18, H19, H24 (see structural formula at the end of Example 9 below) (1S)-(+)-10-Camphorsulfonate 232.20 729.20 230.9495.1497.0727.3 7.57, 7.68, 3.97 maleate 116.07 612.97 114.8495.1497.0 7.45, 7.64, 3.89 cyclamate 179.24 676.14 177.9495.1496.9674.3676.1 7.44, 7.64, 3.89 Phosphate 97.99 594.89 495.1497.0593.1 7.37, 7.61, 3.84 Dimethyl Phosphate 126.05 622.95 124.9495.1497.0621.2623.0 7.50, 7.66, 3.92 p-toluenesulfonate 172.20 669.10 170.9495.1497.0 7.54, 7.71, 3.95 L-lysate 146.19 643.09 145.0495.1497.0 7.36, 7.60, 3.83 L-Lysine Hydrochloride 182.65 679.55 495.1497.0531.1 (HCl) 7.36, 7.60, 3.83 Saccharinate 183.19 680.09 181.9495.1497.0 7.44, 7.64.3.89 mesylate 96.11 593.01 495.1497.0591.2593.1 7.57, 7.68, 3.97 Hydrochloride 36.46 533.36 495.1496.9531.1532.5535.2 7.55, 7.67, 3.95

All salts formed in this process were amorphous.

Example 2

Other amorphous salts of Compound A were prepared using techniques similar to those described in Example 1 above from the following acids:

Hydrobromic acid (1:1 salt)

Hydrochloric acid (1:1 salt)

Sulfuric acid (1:0.5 salt)

1,2-ethanedisulfonic acid (1:0.5 salt)

1S-camphorsulfonic acid (1:1 salt)

(+/-)-camphorsulfonic acid (1:1 salt)

Ethylsulfonic acid (1:1 salt)

Nitric acid (1:1 salt)

Toluenesulfonic acid (1:1 salt)

Methanesulfonic acid (1:1 salt)

p-xylenesulfonic acid (1:1 salt)

2-Mesitylenesulfonic acid (1:1 salt)

1,5-naphthalenesulfonic acid (1:0.5 salt)

Naphthalenesulfonic acid (1:1 salt)

Benzenesulfonic acid (1:1 salt)

Saccharic acid (1:1 salt)

Maleic acid (1:1 salt)

Phosphoric acid (1:1 salt)

D-glutamic acid (1:1 salt)

L-glutamic acid (1:1 salt)

D, L-glutamic acid (1:1 salt)

L-arginine (1:1 salt)

L-lysine (1:1 salt)

L-Lysine hydrochloride (1:1 salt)

Glycine (1:1 salt)

Salicylic acid (1:1 salt)

Tartaric acid (1:1 salt)

Fumaric acid (1:1 salt)

Citric acid (1:1 salt)

L-(-)-malic acid (1:1 salt)

D, L-malic acid (1:1 salt)

D-gluconic acid (1:1 salt)

Embodiment 3: Preparation of amorphous compound A, ethanesulfonic acid salt

Compound A (203 mg; see Preparation A above) was dissolved in ethanol (3 mL), and ethanesulfonic acid (1 eq., 95%, 35 μL) was added to the solution. The mixture was stirred for several minutes, then the solvent was evaporated. The resulting oil was slurried in isooctane and evaporated to dryness until a solid material was obtained. Finally the resulting product was reslurried in isooctane and the solvent was evaporated again to give a white, dry amorphous solid. The solid was dried under vacuum at 40 °C overnight.

Examples 4-9: Preparation of Crystalline Compound A, Esylate Salt

Example 4: Crystallization of an amorphous material

Amorphous Compound A, ethanesulfonate salt (17.8 mg; see Example 3 above) was slurried in methyl isobutyl ketone (600 μL). After 1 week, needle-like crystals were observed, which were filtered and air-dried.

Embodiment 5-7: reaction crystallization (without anti-solvent)

Example 5

Compound A (277 mg; see Preparation A above) was dissolved in methyl isobutyl ketone (3.1 mL). Add ethanesulfonic acid (1 eq., 95%, 48 μL). Precipitation of the amorphous ethanesulfonate salt occurred immediately. Additional methyl isobutyl ketone (6 mL) was added and the slurry was sonicated. Finally a third portion of methyl isobutyl ketone (3.6 mL) was added and the slurry was left under stirring (magnetic stirrer) overnight. The next day, the substance had transformed into needle-like crystals. The slurry was filtered, dried with methyl isobutyl ketone (0.5 mL), and air dried.

Example 6

Compound A (236 mg; see Preparation A above) was dissolved in methyl isobutyl ketone (7 mL) at room temperature. Ethylsulfonic acid (1 eq., 41 μL) was mixed with 2 mL of methyl isobutyl ketone in a bottle. To the solution of Compound A was added as seed crystals crystalline Compound A, ethanesulfonate (see Examples 4 and 5 above). Then 250 μL of ethanesulfonic acid in methyl isobutyl ketone was added in portions over 45 minutes. The solution was again seeded and the temperature was raised to 30°C. Then 500 μL of methyl isobutyl ketone solution was added over about 1 hour. The resulting slurry was left overnight, then the final amount of methyl isobutyl ketone/acid solution was added over 20 minutes. The bottle was washed with 1.5 mL of methyl isobutyl ketone and the wash was added to the slurry. After 6 hours, the crystals were filtered, washed with methyl isobutyl ketone (2 mL), and dried at 40°C under reduced pressure. A total of 258 mg of crystalline salt was obtained, corresponding to a yield of about 87%.

Example 7

Compound A (2.36 g; see Preparation A above) was dissolved in methyl isobutyl ketone (90 mL). To this solution was added compound A, seeds (10 mg) of the ethanesulfonate salt (see Examples 4-6 above), followed by the addition of ethanesulfonic acid (40 [mu]L) in two portions. Additional seeds (12 mg) and two batches of ethanesulfonic acid (2 x 20 μL) were added. The slurry was diluted with methyl isobutyl ketone (15 mL) and the addition of ethanesulfonic acid continued. A total of 330 μL of ethanesulfonic acid was added in portions over 1 hour. A small amount of seed crystals was added and finally the slurry was left under stirring overnight. On the next day, the crystals were filtered off, washed with methyl isobutyl ketone (2 x 6 mL), and dried at 40°C under reduced pressure. After drying, a total of 2.57 g of white crystalline product was obtained, corresponding to a yield of 89%.

Examples 8 and 9: Reactive crystallization (using anti-solvent)

Example 8

Compound A (163 mg; see Preparation A above) was dissolved in isopropanol (1.2 mL). The solution was heated to 35°C. Add ethanesulfonic acid (28 μL). Ethyl acetate (4.8 mL) was then added and the solution was seeded with crystalline Compound A, the ethanesulfonate salt (see Examples 4-7 above). Crystallization started almost immediately. The slurry was held at 35°C for about 80 minutes and then allowed to cool to room temperature (21°C). After 2 hours, the crystals were filtered off, washed 3 times with ethyl acetate (3×0.4 mL), and dried at 40° C. under reduced pressure. In total, 170 mg of the title product were obtained as crystals, corresponding to a yield of about 82%.

Example 9

Compound A (20.0 g; see Preparation A above) was dissolved in isopropanol (146.6 mL) at 40°C and ethanesulfonic acid (3.46 mL, 95%, 1 eq.) was added to the solution. To the resulting clear solution was added compound A, seed crystals of the esylate salt (50 mg; see Examples 4-8 above). Ethyl acetate (234 mL) was then added over 10 minutes. The resulting slightly opaque solution was re-seeded (70 mg) and left under stirring at 40°C for 1 hour to allow crystallization to begin. A total of 352 mL of ethyl acetate was then added at a constant rate over 1 hour. When all the ethyl acetate had been added, the slurry was allowed to stand for 1 hour and then cooled to 21°C over 2 hours. The crystallization was allowed to continue for 1 hour at 21 °C, then filtered off, washed twice with ethyl acetate (50 mL+60 mL), and finally dried under reduced pressure at 40 °C overnight. A total of 21.6 g of white crystalline salt was obtained, corresponding to a yield of about 90%.

Compound A, the esylate salt, was characterized by NMR as follows: 23 mg of the salt was dissolved in deuterated methanol (0.7 mL) trocopy.

A combination of 1D ( ¹ H, ¹³ C and selective NOE) and 2D (gCOSY, gHSQC and gHMBC) NMR experiments was used. All data are in good agreement with the theoretical structure of the salt shown below. There are two conformations of the molecule in methanol. Based on the integration of the peak assigned to H5 (the major conformer) and the peak assigned to H5' (the other conformer), a 70:30 ratio of the two conformers was found. H22 cannot be observed because of the rapid exchange of these protons with the solvent _CD3OD .

The proton and carbon resonances corresponding to position 1 split due to spin coupling with the two fluorine nuclei at this position. The coupling constants are ² J _HF =73 Hz and ¹ J _CF =263 Hz.

¹ H and ¹³ C NMR chemical shift assignments and proton-proton correlations are shown in Table 2.

Table 2 atomic number type ¹³ C displacement/ppm ^{a 1} H shift/ppm ^b and diversity ^c J _HH /Hz 11' CH 117.5 ^e 117.5 ^e 6.90(t)6.88(t) 73( ² J _HF ) twenty two' C 153.5153.5 33' CH 120.0119.7 7.15(s)7.13(s) 44' C 136.2135.9 55' CH 125.0124.9 7.36(s)7.31(s) 66' C 144.5145.3 77' CH 117.3117.2 7.20(s)7.15(s) 88' CH 72.074.0 5.20(s)5.12(s) 99' CO 173.1173.8 1111' CH ₂ 51.649.0 a: 4.38(m)b: 4.21(m)a: 4.06(m)b: 3.99(m) 1212' CH ₂ 21.723.2 a: 2.55(m)b: 2.29(m)a: 2.70(m)b: 2.15(m) 1313' CH 63.166.2 4.80(m)5.22(m) 1414' CO 172.9173.6 1515' NH 8.76(t,br)8.79(t,br) 5.25.2 1616' CH ₂ 43.543.6 4.59 (AB-pattern) 4.46 (AB-pattern) 4.53 (AB-pattern) 4.49 (AB-pattern) 15.915.915.915.9 1717' C 146.9147.0 1818' CH 129.1129.1 7.56(d)7.57(d) 7.87.8 1919' CH 129.2129.4 7.67(d)7.70(d) 7.87.8 2020' C 124.9124.9 2121' C 162.4162.3 twenty two NH ₂ not observed twenty four _CH3 64.8 3.96(s) 101 CH3 1.28(t) 7.4 102 CH2 2.77(m) 7.4

^a Relative to solvent resonance at 49.0 ppm.

^b Relative to solvent resonance at 3.30 ppm.

^c s = singlet, t = triplet, m = multiplet, br = broad, d = doublet

^d Obtained in gCOZY experiments.

^e Resonance is triplet due to coupling with two fluorine nuclei. ¹ J _CF =263 Hz.

_HRMS , calcd. ^{for C24H29ClF2N4O8S(MH)-} _{605.1284 , found 605.1296} .

Compound A, crystalline esylate salt (obtained by one or more of the methods of Examples 4-9 above), was analyzed by XRPD and the results obtained are shown in the table below (Table 3) and in Figure 1 of the accompanying drawings.

table 3 d value () strength(%) strength 16.5 10 m 12.2 74 vs 11.0 4 w 9.0 33 the s 8.3 3 vw 7.6 6 w 6.4 4 w 6.2 12 m 6.0 7 m 5.9 10 m 5.5 15 m 5.4 100 vs 5.1 7 m 4.66 29 the s 4.60 36 the s 4.31 57 the s 4.25 18 m 4.19 20 m 4.13 12 m 4.00 12 m 3.87 13 m 3.83 6 w 3.76 7 m 3.72 6 w 3.57 9 m 3.51 7 m 3.47 5 w 3.39 3 vw 3.31 11 m 3.26 10 m 3.21 8 m 3.16 4 w 3.03 8 m 2.78 4 w 2.74 5 w 2.67 3 vw 2.56 5 w 2.50 5 w 2.46 7 m 2.34 4 w 2.21 5 w 2.00 3 vw 1.98 3 vw

DSC indicated an endotherm with an extrapolated melting onset temperature of about 131 °C. TGA indicated a mass loss of about 0.2% (w/w) around the melting point. The DSC analysis was repeated with a sample of low solvent content, and the results indicated a melting onset temperature of about 144°C.

Example 10: Preparation of Amorphous Compound A, Besylate

Compound A (199 mg; see Preparation A above) was dissolved in ethanol (2 mL). Benzenesulfonic acid (1 eq. 90%, 70 mg) was dissolved in ethanol (1 mL) in a vial. The ethanol solution was added to the compound A solution, the bottle was washed with 1 mL of ethanol, and the wash was added to the mixture. The mixture was stirred for a few minutes, then the ethanol was evaporated until an oil formed. Ethyl acetate (3 mL) was added and the solvent was evaporated to dryness again. An amorphous solid formed.

Example 11-13: Preparation of Compound A, Crystalline Besylate Salt

Example 11: Crystallization of an amorphous material

Amorphous Compound A besylate salt (20.7 mg; see Example 10 above) was slurried in ethyl acetate (600 μL). After 5 days, needle crystals were observed in the slurry.

Examples 12 and 13: Reactive crystallization

Example 12

Compound A (128 mg; see Preparation A above) was dissolved in ethyl acetate (3 mL). To this solution was added the slurry obtained in Example 11 above as seed crystals. Then benzenesulfonic acid (1 eq., 90%, 45 mg) was added. The besylate started to precipitate immediately. Isopropanol (0.8 mL) was added to the slurry and the mixture was again seeded. After two days, the substance had transformed into needle-like crystals. The slurry was filtered, washed with ethyl acetate (3 x 0.2 mL), and dried under vacuum at 40 °C for a short time. In total about 140 mg of white solid was obtained.

Example 13

Compound A (246 mg; see Preparation A above) was dissolved in isopropanol (1.52 mL). Add benzenesulfonic acid (88mg, 90%). Ethyl acetate (3 mL) was added to the clear solution, and the mixture was then seeded to initiate crystallization. After 1 hour, additional ethyl acetate (2.77 mL) was added. Finally the slurry was allowed to crystallize overnight, then the crystals were filtered off, washed with ethyl acetate (3 x 0.3 mL) and dried under vacuum at 40°C. A total of 279 mg of salt was obtained, corresponding to a yield of about 86%.

Compound A, the besylate salt, was characterized by NMR as follows: 20 mg of the salt was dissolved in deuterated methanol (0.7 mL). A combination of 1D ( ¹ H, ¹³ C and selective NOE) and 2D (gCOSY, gHSQC and gHMBC) NMR experiments was used. All data are in good agreement with the theoretical structure of the salt shown below. There are two conformations of the molecule in methanol. Based on the integration of the peak assigned to H12 (the major conformer) and the peak assigned to H12' (the other conformer), a 70:30 ratio of the two conformers was found. H22 cannot be observed because of the rapid exchange of these protons with the solvent _CD3OD .

The proton and carbon resonances corresponding to position 1 split due to spin coupling with the two fluorine nuclei at this position. The coupling constants are ² J _HF =74 Hz and ¹ J _CF =260 Hz.

¹ H and ¹³ C NMR chemical shift assignments and proton-proton correlations are shown in Table 4.

Table 4 atomic number type ¹³ C displacement/ppm ^{a 1} H shift/ppm ^b and diversity ^c J _HH /Hz 11' CH ^{117.5c 117.5c} 6.89(t)6.87(t) 74( ² J _HF ) twenty two' C 153.5153.5 33' CH 120.1119.7 7.15(s)7.12(s) 44' C 136.2135.9 55' CH 125.1124.9 7.35(s)7.31(s) 66' C 144.5145.3 77' CH 117.3117.2 7.20(s)7.14(s) 88' CH 72.874.0 5.20(s)5.12(s) 99' CO 173.1173.8 1111' CH ₂ 51.649.0 a: 4.37(m)b: 4.20(m)a: 4.05(m)b: 3.98(m) 1212' CH ₂ 21.723.2 a: 2.53(m)b: 2.28(m)a: 2.69(m)b: 2.14(m) 1313' CH 63.166.2 4.79(m)5.22(m) 1414' CO 172.9173.6 1515' NH 8.75(t,br)8.78(t,br) 5.35.3 16 CH ₂ 43.5 4.59 (AB-pattern) 4.44 (AB-pattern) 16.0 and 5.216.0 and 4.8 16' 43.6 4.51 (AB-pattern) 4.46 (AB-pattern) 16.016.0 1717' C 146.9147.0 1818' CH 129.2129.2 7.54(d)7.56(d) 8.38.3 1919' CH 129.3129.4 7.66(d)7.69(d) 8.38.3 2020' C 124.9124.9 2121' C 162.4162.4 twenty two NH ₂ not observed twenty four _CH3 64.8 3.95(s) 101 CH 126.9 7.81(m) 102 CH 129.1 7.41(m) 103 CH 131.2 7.42(m) 104 C 146.4

^a Relative to solvent resonance at 49.0 ppm.

^b Relative to solvent resonance at 3.30 ppm.

^c s = singlet, t = triplet, m = multiplet, br = broad, d = doublet

^d Obtained in gCOZY experiments.

^e Resonance is triplet due to coupling with two fluorine nuclei. ¹ J _CF =260 Hz.

^f Difficult to determine connectivity due to overlap between resonances 102 and 103.

_HRMS , calcd _. for _{C28H29ClF2N4O8S} (MH) ^- 653.1284 _, found _653.1312 .

Compound A, crystalline besylate salt (obtained by one or more of the methods of Examples 11-13 above), was analyzed by XRPD and the results obtained are shown in the table below (Table 5) and accompanying drawing 2 .

table 5 d value () strength(%) strength 14.2 12 m 12.6 55 the s 10.2 49 the s 7.5 8 m 6.4 5 w 6.3 30 the s 6.1 5 w 5.9 100 vs 5.7 20 m 5.4 9 m 5.3 11 m 5.1 10 m 4.96 3 vw 4.83 27 the s 4.73 72 vs 4.54 twenty three the s 4.50 10 m 4.35 28 the s 4.30 38 the s 4.24 twenty four the s 4.17 28 the s 4.09 60 vs 4.08 61 vs 3.96 29 the s 3.91 15 m 3.77 twenty two the s 3.62 11 m 3.52 20 m 3.31 44 the s 3.19 8 m 3.15 11 m 3.09 8 m 3.00 7 m 2.89 3 vw 2.86 4 w 2.79 7 m 2.76 6 w 2.72 5 w 2.59 6 w 2.56 9 m 2.54 9 m 2.49 7 m 2.38 8 m 2.16 4 w 2.03 3 vw

DSC indicated an endotherm with an extrapolated melting onset temperature of about 152°C. TGA indicated a mass loss of about 0.1% (w/w) around the melting point.

Example 14: Preparation of amorphous compound A, n-propanesulfonate

Compound A (186 mg; see Preparation A above) was dissolved in isopropanol (1.39 mL) and n-propanesulfonic acid (1 eq., 95%, 39 μL) was added. Ethyl acetate (5.6 mL) was added and the solvent was evaporated to dryness to form an amorphous solid.

Examples 15 and 16: Preparation of Compound A, crystalline n-propanesulfonate salt

Example 15: Crystallization of an amorphous material

Amorphous Compound A, n-propanesulfonate salt (20 mg; see Example 14 above) was dissolved in isopropanol (60 μL) and isopropyl acetate (180 μL) was added. After 3 days, needle crystals were observed.

Embodiment 16: reaction crystallization

Compound A (229 mg; see Preparation A above) was dissolved in isopropanol (1.43 mL). Add n-propanesulfonic acid (1 eq., 95%, 48 μL). Ethyl acetate (2 mL) was added, and the solution was then seeded with the crystalline salt obtained in Example 15 above. Additional ethyl acetate (5 mL) was added and the slurry was left overnight to crystallize. The crystals were filtered off, washed with ethyl acetate (3 x 0.3 mL), and dried under vacuum at 40°C.

Compound A, the n-propanesulfonate salt, was characterized by NMR as follows: 13 mg of the salt was dissolved in deuterated methanol (0.7 mL) trocopy. A combination of 1D ( ¹ H, ¹³ C) and 2D (gCOSY) NMR experiments was used. All data are in good agreement with the theoretical structure of the salt shown below. There are two conformations of the molecule in methanol. Based on the integration of the peak assigned to H12 (the major conformer) and the peak assigned to H12' (the other conformer), a 65:35 ratio of the two conformers was found. H22 cannot be observed because of the rapid exchange of these protons with the solvent _CD3OD .

The proton and carbon resonances corresponding to position 1 split due to spin coupling with the two fluorine nuclei at this position. The coupling constants are ² J _HF =74 Hz and ¹ J _CF =260 Hz.

¹ H and ¹³ C NMR chemical shift assignments and proton-proton correlations are shown in Table 6.

Table 6 atomic number type ¹³ C displacement/ppm ^{a 1} H shift/ppm ^b and diversity ^c J _HH /Hz 11' CH 117.5 ^e 117.5 ^e 6.89(t)6.88(t) 74( ² J _HF ) twenty two' C 153.5153.5 33' CH 120.0119.7 7.16(s)7.13(s) 4 C 136.2 4' 135.9 55' CH 125.1124.9 7.36(s)7.31(s) 66' C 144.5145.3 77' CH 117.3117.2 7.20(s)7.16(s) 88' CH 72.974.1 5.20(s)5.12(s) 99' CO 173.1173.8 1111' CH ₂ 51.649.0 a: 4.37(m)b: 4.20(m)a: 4.06(m)b: 3.98(m) 1212' CH ₂ 21.723.2 a: 2.53(m)b: 2.29(m)a: 2.69(m)b: 2.15(m) 1313' CH 63.166.2 4.80(m)5.22(m) 1414' CO 172.9173.8 1515' NH 8.75(t,br)8.79(t,br) 5.55.5 1616' CH ₂ 43.543.6 4.59 (AB-pattern) 4.45 (AB-pattern) 4.514.50 16.0 and 6.616.0 and 5.3 17 C 146.9 17' 147.0 1818' CH 129.1129.2 7.54(d)7.57(d) 8.58.5 1919' CH 129.2129.4 7.67(d)7.69(d) 8.58.5 2020' C 124.9124.9 2121' C 162.4162.4 twenty two NH ₂ not observed twenty four _CH3 64.7 3.96(s) 101 CH 13.7 1.0(t) 102 CH 19.6 1.78(m) 103 CH 54.6 2.75(m)

^a Relative to solvent resonance at 49.0 ppm.

^b Relative to solvent resonance at 3.30 ppm.

^c s = singlet, t = triplet, m = multiplet, br = broad, d = doublet

^d Obtained in gCOZY experiments.

^e Resonance is triplet due to coupling with two fluorine nuclei. ¹ J _CF =260 Hz.

_{HRMS ,} calcd. for _{C25H31ClF2N4O8S} (MH) ^- _619.1441 , found _619.1436 .

Compound A, the crystalline n-propanesulfonate salt (obtained by one or more of the methods of Examples 15 and 16 above), was analyzed by XRPD and the results are shown in the table below (Table 7) and accompanying Figure 3 .

Table 7 d value () strength(%) strength 14.0 4 w 12.4 87 vs 10.0 30 the s 8.0 3 vw 7.5 7 m 7.0 0.6 vw 6.7 1 vw 6.4 1 vw 6.2 12 m 6.1 3 vw 5.8 100 vs 5.7 11 m 5.5 3 vw 5.4 5 w 5.3 5 w 5.2 2 vw 5.1 3 vw 4.94 3 vw 4.78 twenty one the s 4.68 42 the s 4.51 10 m 4.49 7 m 4.40 5 w 4.32 10 m 4.29 10 m 4.25 twenty two the s 4.19 14 m 4.14 15 m 4.07 twenty three the s 4.04 20 m 3.94 16 m 3.88 10 m 3.73 15 m 3.65 2 vw 3.59 3 vw 3.48 18 m 3.28 twenty three m 3.12 4 w 3.06 3 vw 2.97 6 w 2.84 2 vw 2.81 3 vw 2.76 2 vw 2.73 3 vw 2.70 2 vw 2.57 2 vw 2.54 6 w 2.51 6 w 2.46 8 m 2.42 2 vw 2.39 3 vw 2.36 3 vw 2.32 2 vw 2.14 3 vw 2.01 2 vw

DSC indicated an endotherm with an extrapolated melting onset temperature of about 135°C. TGA showed no loss of mass around the melting point.

Example 17

Example 17-A: Preparation of Amorphous Compound A n-Butanesulfonate

Amorphous compound A (277 mg) was dissolved in IPA (1.77 ml), and butanesulfonic acid (about 1 eq. 70 μL) was added. Ethyl acetate (6ml) was added and the solvent was evaporated to dryness to form an amorphous solid.

Example 17-B: Preparation of compound A butanesulfonate crystals

Amorphous Compound A butanesulfonate (71.5 mg; see preparation above) was slurried in ethyl acetate (500 μl) overnight. The crystals were filtered off and air dried.

Compound A, the butane sulfonate salt, was characterized by NMR as follows: 21.6 mg of the salt was dissolved in deuterated dimethyl sulfoxide (0.7 ml) and determined by ¹ H and ¹³ C NMR spectroscopy. Its spectrum is very similar to other salts of the same compound and is in good agreement with the structure shown below. Most of the resonances in the spectrum appear as groups of two peaks due to the slow rotation along the C9-N10 bond, which results in the simultaneous presence of both atropisomers in this solution. Other salts of the same compound also show this phenomenon.

The two fluorine nuclei at position 1 split the proton and carbon resonances at this position. The coupling constants are ² J _HF =73 Hz and ¹ J _CF =258 Hz.

Table 1 gives the chemical shifts of protons and carbons. Protons at positions 22 and 24 were not detected due to chemical exchange. In the proton spectrum corresponding to these protons, there are very broad peaks between 8 and 9 ppm.

Table 8

¹ H and ¹³ C NMR chemical shift assignments of Compound A n-butanesulfonate salt in deuterated dimethyl sulfoxide at 25°C. atomic number type ¹³ C displacement/ppm ^{a 1} H shift/ppm ^b and diversity ^c J _HH /Hz 11' CHF ₂ 116.3 ^d 116.3 ^d 7.29(t)7.28(t) 73( ² J _HF )73( ² J _HF ) twenty two' C 151.5151.3 nana nana 33' CH 118.0117.6 7.25(t) ^e 7.21(t) ^e ndnd 44' C 133.8133.4 nana nana 55' CH 123.8123.6 7.34(t) ^e 7.25(t) ^e ndnd 66' C 144.5145.2 nana nana 77' CH 116.3116.1 7.19(t) ^e 7.12(t) ^e ndnd 88' CH 70.971.2 5.13(s)4.99(s) nana 99' CO 170.6171.1 nana nana 1111' CH ₂ 50.046.9 a: 4.24(m)b: 4.12(m)3.85(m) ndnd 1212' CH ₂ 20.521.7 a: 2.41(m)b: 2.10(m)a: 2.60(m)b: 2.02(m) ndnd 1313' CH 61.263.9 4.65(dd)5.12(m) 5.6 and 8.9nd 1414' CO 170.2171.0 nana nana 1616' CH ₂ 41.842.0 4.38(m)4.38(m) ndnd 17 C 144.7 na na 18 CH 127.5127.6 7.44(d)7.44 8.2nd 19 CH 127.8 7.66(d) 8.2 20 C 125.1 na na twenty one C 157.9 na na 2424' _CH3 63.363.3 3.83(s)3.82(s) nana 26 CH ₂ 51.4 2.41(m) nd 27 CH ₂ 27.3 1.52(m) nd 28 CH ₂ 21.7 1.30(m) nd 29 _CH3 14.0 0.83(t) 7.3

^a Relative to solvent resonance at 49.0 ppm.

^b Relative to solvent resonance at 3.30 ppm.

^c s = singlet, d = doublet, dd = double doublet, t = triplet, m = multiplet.

^d Resonance is triplet due to coupling with two fluorine nuclei F1. ¹ J _CF = 258 Hz.

^e The ⁴ J _HH coupling to the meta proton is not fully resolved.

na = not available, nd = not determined

HRMS, Calcd. for C ₂₆ H ₃₂ ClF ₂ N ₄ O ₈ S(MH) ^- 633.1597, found 633.1600

The crystals of compound A n-butanesulfonate salt (obtained in Example 17-B as described above) were analyzed by XRPD, and the results are shown in the following table (Table 9) and accompanying drawing 4 .

Table 9 d value () strength(%) strength 14.3 8 m 12.8 81 vs 10.3 44 the s 8.2 4 w 7.7 13 m 6.7 2 vw 6.4 8 m 6.2 18 m 6.0 100 vs 5.8 29 the s 5.6 4 w 5.4 11 m 5.3 16 m 5.1 15 m 4.98 6.5 w 4.91 34 the s 4.76 56 the s 4.57 20 m 4.42 13 m 4.36 19 m 4.30 45 the s 4.18 42 the s 4.13 88 vs 4.01 34 the s 3.92 28 the s 3.82 18 m 3.64 6.6 w 3.58 16 m 3.47 5 w 3.44 6 w 3.38 12 m 3.35 32 the s 3.32 twenty two the s 3.29 12 m 3.20 8 m 3.17 9 m 3.02 12 m 2.90 6 w 2.81 3.9 vw 2.75 3 vw 2.64 3.5 vw 2.59 10 m 2.57 8 m 2.50 4 w 2.45 5 w 2.40 6 w 2.31 3 vw

DSC indicated an endotherm with an extrapolated melting onset temperature of about 118°C, and TGA indicated a weight loss of about 0.04% at 25-150°C.

Example 18: Preparation of Salt of Compound B

Example 18-A: General method for preparing salts

The following general procedure was used to prepare the salt of Compound B: 200 mg of Compound B (see Preparation B above) was dissolved in 5 mL of MIBK (methyl isobutyl ketone). To this solution was added a solution of the corresponding acid (1.0 or 0.5 molar equivalents, as indicated in Table 10) dissolved in 1.0 mL of MIBK. After stirring at room temperature for 10 minutes, the solvent was removed by rotary evaporator. The remaining solid material was redissolved in about 8 mL of acetonitrile: _H2O (1:1). Freeze-drying yielded colorless amorphous products, respectively.

Acid used:

Ethylsulfonate (ethanesulfonic acid)

Benzenesulfonate (Benzenesulfonic Acid)

cyclamate

Sulfate

bromide

p-toluenesulfonate

2-naphthalenesulfonate

hemisulfate

mesylate

Nitrate

Hydrochloride

Appropriate characteristic data are shown in Table 10

Table 10 Salt Mw acid Mw salt MS ES- ethanesulfonate 110.13 643.01 108.8531.1641.0 Besylate 158.18 691.06 156.8531.1689.2 cyclamate 179.24 712.12 177.9531.2710.4 Sulfate 98.08 630.96 531.1 bromide 80.91 613.79 531.2613.1 p-toluenesulfonate 172.20 705.08 170.9531.1703.1 2-naphthalenesulfonate 208.24 741.12 206.9531.1739.3 hemisulfate 98.07 1163.8(1:2)630.85(1:1) 531.1631.0 mesylate 96.11 628.99 531.1627.1 Nitrate 63.01 595.89 531.0594.0 Hydrochloride 36.46 569.34 531.0569.0

All salts formed in this example were amorphous.

Example 18-B

Other amorphous salts of Compound B were prepared from the following acids using techniques similar to those described in Example 18-A above:

1,2-ethanedisulfonic acid (0.5 salt)

1S-camphorsulfonic acid

(+/-)-camphorsulfonic acid

p-xylenesulfonic acid

2-Mesitylenesulfonic acid

Saccharinic acid

maleic acid

phosphoric acid

D-glutamic acid

L-Arginine

L-Lysine

L-Lysine*HCl

Example 18-C: Preparation of Amorphous Compound B, Hemi-1,5-Naphthalene Disulfonate

Amorphous Compound B (110.9 mg) was dissolved in 2.5 mL 2-propanol, and 0.5 equivalents of 1,5-naphthalene-disulfonic acid tetrahydrate (dissolved in 1 mL 2-propanol) was added. The sample was stirred overnight. Only small particles (amorphous) or oil droplets were observed by microscopy. The sample was evaporated to dryness.

Example 18-D: Preparation of compound B, crystalline hemi-1,5-naphthalene disulfonate

Crystallization experiments were performed at room temperature. Amorphous compound B (0.4 g) was dissolved in ethanol (1.5 mL), and 0.5 equivalents of 1,5-naphthalene-disulfonic acid tetrahydrate (1.35 g, 10% mixture in ethanol) was added. Heptane (0.7 mL) was then added until the solution became slightly cloudy. After about 15 minutes, the solution became cloudy. After about 30 minutes, a thin slurry was obtained and additional heptane (1.3 mL) was added. The slurry was then left overnight to mature. To dilute the thick slurry, a mixture of ethanol and heptane (1.5 mL and 1.0 mL, respectively) was added. After about 1 hour, the slurry was filtered and the crystals were washed successively with a mixture of ethanol and heptane (1.5:1) and then pure heptane. The crystals were dried at room temperature for 1 day. The dried crystals weighed 0.395 g.

Example 18-E: Preparation of compound B, hemi-1,5-naphthalene disulfonate crystals

Amorphous Compound B (1.009 g) was dissolved in 20 mL 2-propanol + 20 mL ethyl acetate. 351.7 mg of 1,5-naphthalene-disulfonic acid tetrahydrate dissolved in 20 mL of 2-propanol was added dropwise. A precipitate started to form within about 5 minutes. The slurry was stirred overnight, then filtered.

Example 18-F: Preparation of Compound B, Crystalline Hemi-1,5-Naphthalene Disulfonate

Dissolve 430.7 mg of 1,5-naphthalene disulfonate in 30 mL of 1-propanol. The solution was heated to boil to dissolve the material. The solution was left at room temperature overnight to crystallize, and the crystals were filtered.

Example 18-G: Preparation of compound B, hemi-1,5-naphthalene disulfonate crystals

The mother liquor from Example 18-F was evaporated and the solid residue (61.2 mg) was dissolved in 6 mL of acetonitrile/1-propanol, ratio 2:1. The solution was left at room temperature overnight to crystallize, and the crystals were filtered.

Example 18-H: Preparation of Compound B, Crystalline Hemi-1,5-Naphthalene Disulfonate

The sample from Example 18-C was dissolved in about 2 mL of methanol. Ethanol (approximately 3 mL) was added as an anti-solvent at room temperature and seeded. Crystallization did not occur, so the solvent was evaporated (about half the amount), and a portion of ethanol (about 2 mL) and seed crystals were added. When stirred overnight at room temperature, crystalline particles formed.

Example 18-I: Preparation of compound B, hemi-1,5-naphthalene disulfonate crystals

Amorphous compound B (104.1 mg) was dissolved in 2-propanol, and 1 equivalent of 1,5-naphthalene-disulfonic acid tetrahydrate dissolved in 2-propanol was added. The amount of 2-propanol was about 2.5 mL in total. The solution was stirred at 44°C for about 80 minutes and a precipitate formed. According to polarized light microscopy, the particles crystallized. Filter the sample.

Example 18-J: Preparation of compound B, crystalline hemi-1,5-naphthalene disulfonate

Compound B, hemi-1,5-naphthalene disulfonate (56.4 mg) was dissolved in 1.5 mL of methanol. Methyl ethyl ketone (3 mL) was added. Seed crystals were added to the solution and crystallization began. The crystals were filtered, washed with methyl ethyl ketone, and air dried.

Example 18-K: Preparation of Compound B, Crystalline Hemi-1,5-Naphthalene Disulfonate

Amorphous Compound B (161.0 mg) was dissolved in 3.5 mL of 1-butanol, and the solution was heated to 40°C. In another beaker, dissolve 57.4 mg of naphthalene-disulfonic acid tetrahydrate in 3 mL of 1-butanol. Two drops of this acid solution were added to the compound B solution. The solution was then seeded and after 2 hours the rest of the acid solution (at 40°C) was added slowly. The temperature was then slowly lowered to room temperature and the assay was left overnight with stirring. The slurry was filtered, washed with 1-butanol, and dried under vacuum at 44°C for 2 hours. The yield was 83%.

characterize

Compound B, crystalline hemi-1,5-naphthalene disulfonate salt obtained by Example 18-D, was determined via NMR as follows:

21.3 mg of the salt was dissolved in deuterated methanol, and 0.7 ml was taken for NMR spectroscopy. A combination of 1D ( ¹ H, ¹³ C and selective NOE) and 2D (gCOSY, gHSQC and gHMBC) NMR experiments was used. All data are in good agreement with the proposed structure shown below. Assign all carbons and the protons attached to them. Protons attached to heteroatoms were exchanged for deuterium from the solvent and were not detected. Most resonances in ^{1D1H and13C} NMR spectra appear as groups of two peaks. This is due to the slow rotation along the C9-N10 bond, which results in the simultaneous presence of both atropisomers in the solution. 1D NOE experiments confirmed this. When the resonance of one atropisomer is illuminated, the saturation is shifted to the corresponding peak of the other atropisomer. The resonance corresponding to the 1,5-naphthalene disulfonate counterion does not exhibit rotational enantiomerism.

There are 4 fluorine atoms in the molecule. They split certain proton and carbon resonances. The proton and carbon resonances corresponding to position 1 split due to spin coupling with the two fluorine nuclei at this position. The coupling constants are ² J _HF =73 Hz and ¹ J _CF =263 Hz. Furthermore, the proton resonance corresponding to H19 is a distorted doublet with ³ _JHF = 6.9 Hz due to spin coupling with the 18-position fluorine nucleus. The carbon resonances corresponding to C17, C18, C19 and C20 also exhibit coupling to these fluorine nuclei. The C17 and C20 resonances are triplet with ² J _CF = 19 Hz and ³ J _CF = 11 Hz, respectively. The C18 resonance has a double doublet with coupling constants ¹ J _CF = 251 Hz and ³ J _CF = 8 Hz. The C19 resonance is a multiplet.

Comparing the magnitude of the resonance integral corresponding to the 1,5-naphthalene disulfonate counter ion and the parent compound, the stoichiometric relationship between a 1,5-naphthalene disulfonate counter ion and the molecular crystallization of the two parent compounds is given.

¹ H and ¹³ C NMR chemical shift assignments and proton-proton correlations are shown in Table 11.

Table 11 atomic number type ¹³ C displacement/ppm ^{a 1} H shift/ppm ^b and diversity ^c J _HH /Hz Associated with ¹ H ^d via bond 11' CHF2 117.5 ^e 117.5 ^e 6.91(t)6.87(t) 73( ² J _HF )73( ² J _HF ) ndnd twenty two' C 153.5153.3 nana nana nana 33' CH 120.0119.6 7.14(t) ⁿ 7.11(t) ⁿ ndnd 5, 75', 7' 44' C 136.1135.8 nana nana nana 55' CH 125.0124.9 7.31(t) ⁿ 7.28(t) ⁿ ndnd 3, 73', 7' 66' C 144.4145.3 nana nana nana 77' CH 117.2117.1 7.16(t) ⁿ 7.12(t) ⁿ ndnd 3, 53', 5' 88' CH 72.973.6 5.15(s)5.07(s) nana ndnd 99' CO 173.0173.5 nana nana nana 11 CH ₂ 51.5 a: 4.29(m) b: 4.13 nd 12, 13 11' 48.6 (m)a: 4.01(m)b: 3.93(m) nd 12', 13' 1212' CH ₂ 21.722.8 a: 2.46(m)b: 2.17(m)a: 2.61(m)b: 2.03(m) ndnd 11, 1311', 13' 1313' CH 62.865.8 4.70(dd)5.14(dd) 6.0 and 9.45.6 and 9.1 1212' 1414' CO 172.4173.2 nana nana nana 1616' CH ₂ 32.332.5 4.51(m)4.51(m) ndnd ndnd 17 C 121.0 ^f na na na 18 CF ^162.8g na na na 19 CH 112.7l 7.35(d) 6.9( ³ J _HF ) nd 20 C 127.9k na na na 2121' C 160.0159.9 nana nana nana 2424' _CH3 64.864.8 3.93(s)3.92(s) nana ndnd 25 C 142.4 na na na 26 CH 126.8 8.16(d) 7.2 27, 28 27 CH 125.9 7.54(dd) 8.6 and 7.2 26, 28 28 CH 131.0 8.97(d) 8.6 26, 27 29 C 131.1 na na na

^a Relative to solvent resonance at 49.0 ppm.

^b Relative to solvent resonance at 3.30 ppm.

^c s = singlet, d = doublet, dd = double doublet, t = triplet, m = multiplet.

^d Obtained in gCOZY experiments.

^e Resonance is triplet due to coupling with two fluorine nuclei F1. ¹ J _CF =263 Hz.

^f Resonance is triplet due to coupling with two fluorine nuclei F18. ² J _CF = 19 Hz.

^g Resonance is double doublet due to coupling with two fluorine nuclei F18. ¹ J _CF =251 Hz and ³ J _CF =8 Hz.

ⁱ Resonance is multiplet due to coupling to two fluorine nuclei F18.

^{The k} resonance is a triplet due to coupling with two fluorine nuclei F18. ³ J _CF = 11 Hz.

The ⁴ J _HH coupling of ⁿ to the meta proton is not fully resolved.

na = not available, nd = not determined

Crystals of compound B, hemi-1,5-naphthalene disulfonate salt (obtained by Example 18-I above) were analyzed by XRPD and the results are shown in the table below (Table 12) and accompanying drawing 5 .

Table 12 d value () strength(%) strength 18.3 99 vs 12.5 twenty two the s 9.9 twenty two the s 9.1 67 vs 8.0 18 m 7.5 17 m 6.8 37 the s 6.7 59 the s 6.1 39 the s 6.0 twenty one the s 5.6 66 vs 5.5 98 vs 4.94 48 the s 4.56 59 the s 4.39 35 the s 4.27 33 the s 4.13 81 vs 4.02 87 vs 3.86 88 vs 3.69 69 vs 3.63 100 vs 3.57 49 the s 3.48 53 the s 3.23 35 the s 3.19 43 the s 3.16 38 the s

DSC indicated an endotherm with an extrapolated melting onset temperature of about 183°C, and TGA indicated a 0.3% weight loss at 25-110°C.

Example 19: Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-(S)Aze-Pab(OMe)besylate

N-Methylmorpholine: NMM

O-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate: TBTU

To a stirred solution of Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)OH (12.6g, 50mmol) in ethyl acetate (126ml) at 0°C N-methylmorpholine (16.5ml, 150mmol), HAze-Pab(OMe).2HCl (16.8g, 50mmol; see compound C(vi) description herein) and TBTU (16.7, 50mmol) were added. The reaction was allowed to warm to room temperature and stirred overnight. The reaction mixture was washed with water, 15% w/v potassium carbonate solution, water, brine and water, dried and concentrated.

To this partially concentrated solution was added ethyl acetate (115ml) at 40°C, and a solution of benzenesulfonic acid (7.1g, 45mmol) in 2-propanol (38.4ml) was added. The solution was seeded and stirred at 40°C for 2 hours, then at room temperature overnight. When the precipitation of the besylate salt was complete, the product was filtered, washed and dried under vacuum at 40°C to afford the subtitle compound (22.6 g, 69%).

Example 20: Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-(S)Aze-Pab(2,6-diF)(OMe)

The coupling reaction of Example 19 can be repeated using HAze-Pab(2,6-diF)(OMe) to precipitate the end product such as hemi-1,5-naphthalenedisulfonate.

To a solution of Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)OH (10.6 g, 42 mmol) in ethyl acetate (66 ml) was added N- Methylmorpholine (6.2 g, 61.2 mmol), HAze-Pab(2,6-diF)(OMe) (conveniently prepared from Cpd B prep. (xi), 12.0 g, 38.2 mmol) and TBTU (15.3 g, 48 mmol). The reaction was allowed to warm to room temperature and stirred overnight. The reaction mixture was washed with water, 15% w/v potassium carbonate solution (x2), water, brine and water, dried and partially concentrated. The solution was dried over anhydrous sodium sulfate (24 g), filtered through the desiccant, and the filtrate was concentrated to obtain a foam (12.2 g, 60%).

abbreviation

Ac = acetyl

APCI = Atmospheric Pressure Chemical Ionization (for MS)

API = Atmospheric Pressure Ionization (for MS)

aq. = aqueous

Aze (&(S)-Aze) = (S)-azetidine-2-carboxylate (unless otherwise stated)

Boc = tert-butoxycarbonyl

br = broad peak (for NMR)

CI = chemical ionization (for MS)

d = days

d = doublet (for NMR)

DCC = Dicyclohexylcarbodiimide

dd = double doublet (for NMR)

DIBAL-H = diisobutylaluminum hydride

DIPEA = Diisopropylethylamine

DMAP=4-(N,N-dimethylamino)pyridine

DMF=N,N-Dimethylformamide

DMSO = dimethyl sulfoxide

DSC = Differential Scanning Calorimetry

DVT = deep vein thrombosis

EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

eq. = equivalent

ES = electrospray

ESI = electrospray interface

Et = ethyl

Ether (ether) = ether

EtOAc = ethyl acetate

EtOH = ethanol

Et ₂ O = diethyl ether

FT-IR = Fourier Transform Infrared Spectroscopy

gCOSY = gradient selectivity correlation chromatography

gHMBC = Gradient Selective Heterokaryotic Multiple Correlation Chromatography

gHSQC = gradient selective heterokaryon single quantum cohesion

HATU=O-(azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

HBTU=[N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium hexafluorophosphate]

HCl = hydrochloric acid, hydrogen chloride gas or hydrochloride (depending on context)

Hex = hexane

HOAc = acetic acid

HPLC = high performance liquid chromatography

LC = liquid chromatography

m = multiplet (for NMR)

Me = methyl

MeOH = Methanol

min.=minute

MS = mass spectrometry

MTBE = methyl tert-butyl ether

NMR = nuclear magnetic resonance

OAc = acetate

Pab = p-amidinobenzylamino

H-Pab = p-Amidinobenzylamine

Pd/C = palladium on carbon

Ph = phenyl

PyBOP = (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate

q = quartet (for NMR)

QF = Tetrabutylammonium fluoride

rt/RT = room temperature

s = singlet (for NMR)

t = triplet (for NMR)

TBTU=[N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)urea tetrafluoroborate]

TEA = triethylamine

Teoc=2-(trimethylsilyl)ethoxycarbonyl

TEMPO = 2,2,6,6-tetramethyl-1-piperidinyloxy radical

TFA = trifluoroacetic acid

TGA = thermogravimetric analysis

THF = Tetrahydrofuran

TLC = Thin Layer Chromatography

UV = Ultraviolet

The prefixes n-, s-, i-, t- and tert- have their usual meanings: normal-, primary-, iso- and tert-.

Certain aspects of the invention are provided as follows:

When aspects refer to other aspects, that reference also includes sub-aspects. For example, reference to aspect 14 refers to aspects 14 and 14A.

1. A pharmaceutically acceptable acid addition salt of a compound of formula I,

in

R ¹ represents C _1-2 alkyl substituted by one or more fluorine substituents;

R ² represents C _1-2 alkyl; and

n represents 0, 1 or 2.

2. A compound as described in aspect 1, wherein the acid is an organic acid.

3. A compound as described in aspect 1, wherein the acid is a sulfonic acid.

4. A compound as described in aspect 3, wherein the acid is 1,2-ethanedisulfonic acid, camphorsulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, pentanesulfonic acid, toluenesulfonic acid , methanesulfonic acid, p-xylenesulfonic acid, 2-mesitylenesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, hydroxybenzenesulfonic acid, 2-hydroxyethanesulfonic acid or 3-hydroxyethanesulfonic acid.

5. A compound as described in aspect 3, wherein the acid is a C _1-6 alkanesulfonic acid or an optionally substituted arylsulfonic acid or an optionally substituted aryldisulfonic acid.

6. The compound as described in aspect 4 or aspect 5, wherein the acid is ethanesulfonic acid, n-propanesulfonic acid or benzenesulfonic acid.

6A. A compound as described in aspect 4 or aspect 5, wherein the acid is ethanesulfonic acid, n-propanesulfonic acid, benzenesulfonic acid, 1,5-naphthalenedisulfonic acid or n-butanesulfonic acid.

7. The compound as described in any one of aspects 1-6, wherein R ¹ represents —OCHF ₂ or —OCH ₂ CH ₂ F.

8. Compounds as described in any one of aspects 1-7, wherein R ² represents methyl.

9. The compound as described in any one of aspects 1-8, wherein n represents 0 or 2.

10. The compound as described in aspect 9, wherein n represents 2 and the two fluorine atoms are located in two ortho positions relative to the point of attachment of the benzene ring to the -NH- _CH2- group.

11. The compound as described in any one of aspects 1-10, wherein the compound of formula I is Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-(S) Aze-Pab (OMe).

12. The compound as described in any one of aspects 1-10, wherein the compound of formula I is Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O)-(S) Aze-Pab(2,6-diF)(OMe).

13. The compound as described in any one of aspects 1-10, wherein the compound of formula I is Ph(3-Cl)(5- _OCH2CH2F )-(R)CH( _OH )C(O)- (S)Aze-Pab(OMe).

14. The compound as described in any one of aspects 1-13, wherein said compound is in substantially crystalline form.

14A. The compound as described in any one of aspects 1-13, wherein said compound is in partially crystalline form.

15. A compound as described in any one of aspects 1-9, 11 or 14, wherein said compound is Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)-( S) Aze-Pab (OMe), ethanesulfonate.

16. The compound as described in aspect 15, characterized by a differential scanning calorimetry curve, wherein the differential scanning calorimetry is carried out in a closed pan with pinholes at a heating rate of 10° C./min under nitrogen flow, and exhibit an endothermic phenomenon with an extrapolated onset temperature of about 131° C.; and/or its X-ray powder diffraction pattern is characterized by having a temperature at 16.5, 12.2, 9.0, 7.6, 6.2, 6.0, 5.9, 5.5, 5.4, d - a peak of value, and/or substantially as defined in Table 3 and/or accompanying drawing 1.

16A. The compound as described in aspect 16, characterized in that its X-ray powder diffraction pattern is characterized by peaks with strong and very strong intensities as defined in Table 3.

17. A compound as described in any one of aspects 1-9, 11 or 14, wherein said compound is Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)-( S) Aze-Pab (OMe), besylate.

18. The compound as described in aspect 17, characterized by a differential scanning calorimetry curve, wherein the differential scanning calorimetry is carried out in a closed pan with pinholes at a heating rate of 10° C./min under nitrogen flow, and exhibit an endothermic phenomenon with an extrapolated onset temperature of about 152° C.; and/or its X-ray powder diffraction pattern is characterized by having a temperature at 14.2, 12.6, 10.2, 7.5, 6.4, 6.3, 6.1, 5.9, 5.7, 5.4, 5.3, 5.1, 4.83, 4.73, 4.54, 4.50, 4.35, 4.30, 4.24, 4.17, 4.09, 4.08, 3.96, 3.91, 3.77, 3.62, 3.52, 3.31, 3.19, 3.15, 3.09, 3.00, 2.79, 2.76, Peaks with d-values of 2.72, 2.59, 2.56, 2.54, 2.49 and 2.38A, and/or substantially as defined in Table 5 and/or Figure 2 of the accompanying drawings.

18A. The compound as described in aspect 18, characterized in that its X-ray powder diffraction pattern is characterized by peaks with strong and very strong intensities as defined in Table 5.

19. A compound as described in any one of aspects 1-9, 11 or 14, wherein said compound is Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O)-( S) Aze-Pab (OMe), n-propanesulfonate.

20. The compound as described in aspect 19, characterized by a differential scanning calorimetry curve, wherein the differential scanning calorimetry is carried out in a closed pan with pinholes at a heating rate of 10° C./min under nitrogen flow, and exhibit an endothermic phenomenon with an extrapolated onset temperature of about 135°C; and/or its X-ray powder diffraction pattern is characterized by having a temperature at 12.4, 10.0, 7.5, 6.2, 5.8, 5.7, 5.4, 5.3, 4.78, Peaks with d-values of 4.68, 4.51, 4.49, 4.40, 4.32, 4.29, 4.25, 4.19, 4.14, 4.07, 4.04, 3.94, 3.88, 3.73, 3.48, 3.28, 2.97, 2.54, 2.51 and 2.46, and/or substantially As defined above in Table 7 and/or accompanying drawing 3.

20A. The compound as described in aspect 20, characterized in that its X-ray powder diffraction pattern is characterized by peaks with strong and very strong intensities as defined in Table 7. 20A.

20B. A compound as described in any one of aspects 1-10, 12, 14 or 14A, wherein said compound is of formula Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C(O )-(S)Aze-Pab(2,6-diF)(OMe), hemi-1,5-naphthalenedisulfonate.

20C. The compound as described in aspect 20B, characterized in that its X-ray powder diffraction pattern is characterized by peaks with strong and very strong intensities as defined in Table 12.

21. A process for the preparation of a compound as described in any one of aspects 1-20, said process comprising adding an acid to a compound of formula I as described in aspect 1.

22. A process for the preparation of a compound as described in aspect 14 or any one of aspects 15-20 (as dependent on aspect 14), which process comprises crystallizing a compound as described in any one of aspects 1-13.

23. A process for the preparation of a compound as described in aspect 14 or any one of aspects 15-20 (as dependent on aspect 14), comprising carrying out the process as described in aspect 21 followed by the process as described in aspect 22 method.

24. A method as described in aspect 22 or aspect 23, comprising crystallizing said compound from a solvent.

25. The method as described in aspect 24, wherein the solvent is selected from lower alkyl acetates, lower alkyl alcohols, lower dialkyl ketones, aliphatic hydrocarbons and aromatic hydrocarbons.

26. The method as described in aspect 24, which comprises dissolving the compound as described in aspect 1 in amorphous form in a solution selected from the group consisting of lower alkyl alcohols, lower alkyl acetates, lower dialkyl ketones, and in the solvent of the mixture, and then crystallized.

27. A method as described in aspect 26, said method comprising:

(a) dissolving said compound in lower alkyl alcohol, then adding lower alkyl acetate or lower dialkyl ketone; or

(b) The compound is dissolved in a mixture of a lower alkyl alcohol and a lower alkyl acetate or a mixture of a lower alkyl alcohol and a lower dialkyl ketone.

28. The method as described in aspect 27, wherein the solvent is selected from the group consisting of methyl isobutyl ketone, isopropanol, ethyl acetate, isopropyl acetate and mixtures thereof.

29. A method as described in aspect 24, which comprises carrying out the method as described in aspect 21, and then dissolving the formed compound from a solvent system comprising lower alkyl acetate, lower dialkyl ketone or hydrocarbon direct crystallization.

30. The method as described in aspect 29, wherein the solvent system is selected from the group consisting of: isopropanol, isopropyl acetate, n-butyl acetate, toluene, methyl isobutyl ketone, ethyl acetate and mixtures thereof.

31. A method as described in aspect 24, which comprises preforming a compound of formula I in a lower alkyl alcohol, followed by addition of a lower alkyl acetate, lower dialkyl ketone or hydrocarbon.

31A. The method as described in any one of aspects 25-31, wherein the term lower alkyl refers to linear or branched (1-4C)alkyl.

32. The method as described in aspect 31, wherein the solvent is selected from the group consisting of methanol, ethanol, isopropanol, methyl isobutyl ketone, n-butyl acetate, toluene, isooctane, n-heptane, ethyl acetate esters and isopropyl acetate.

33. A process for the preparation of a crystalline compound as defined in aspect 15 or aspect 16, said process comprising slurrying a pre-formed salt in a mixture of methyl isobutyl ketone or isopropanol and ethyl acetate.

34. A process for the preparation of a crystalline compound as defined in aspect 15 or aspect 16, comprising adding ethanesulfonic acid (optionally in the form of a solution in methyl isobutyl ketone) to Ph(3-Cl )(5- _OCHF2 )-(R)CH(OH)C(O)-(S)Aze-Pab(OMe) in solution in methyl isobutyl ketone.

35. A process for the preparation of a crystalline compound as described in aspect 15 or aspect 16, said process comprising adding ethanesulfonic acid to Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C( A solution of O)-(S)Aze-Pab(OMe) in isopropanol was then added with ethyl acetate as anti-solvent.

36. A process for the preparation of a crystalline compound as defined in aspect 17 or aspect 18, said process comprising slurrying a pre-formed salt in ethyl acetate, methyl isobutyl ketone or isopropyl acetate.

37. A process for the preparation of a crystalline compound as defined in aspect 17 or aspect 18, comprising adding benzenesulfonate to Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C(O )-(S)Aze-Pab(OMe) in ethyl acetate, followed by addition of isopropanol to facilitate crystallization.

38. A process for the preparation of a crystalline compound as defined in aspect 17 or aspect 18, said process comprising adding benzenesulfonic acid to Ph(3-Cl)(5-OCHF ₂ )-(R)CH(OH)C( A solution of O)-(S)Aze-Pab(OMe) in isopropanol was then added with ethyl acetate as anti-solvent.

39. A process for the preparation of a crystalline compound as defined in aspect 19 or aspect 20, said process comprising slurrying a pre-formed salt in a mixture of isopropanol and isopropyl acetate or a mixture of isopropanol and ethyl acetate change.

40. A process for the preparation of a crystalline compound as defined in aspect 19 or aspect 20, said process comprising adding n-propanesulfonic acid to Ph(3-Cl)(5- _OCHF2 )-(R)CH(OH)C A solution of (O)-(S)Aze-Pab(OMe) in isopropanol was then added with ethyl acetate or isopropyl acetate as anti-solvent.

41. Compound obtainable by a method according to any one of aspects 21-40.

42. A compound as described in any one of aspects 1-20 or 41 for use as a medicament.

43. A pharmaceutical formulation comprising a compound as defined in any one of aspects 1-20 or 41 in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

44. A compound as defined in any one of aspects 1-20 or 41 of a medicament, or a pharmaceutically acceptable derivative thereof.

45. A compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, for use in the treatment of a condition in which inhibition of thrombin is required.

46. A compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, for use in the treatment of a condition for which anticoagulation therapy is indicated.

47. A compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, for use in the treatment of thrombosis.

48. A compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, for use as an anticoagulant.

49. Use of a compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for the treatment of a condition in which inhibition of thrombin is required.

50. Use of a compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, for the manufacture of a medicament for the treatment of a condition for which anticoagulation therapy is indicated.

51. The use as described in aspect 49 or aspect 50, wherein the condition is thrombosis.

52. The use as described in aspect 49 or aspect 50, wherein the condition is hypercoagulability in blood and/or tissue.

53. Use of a compound as defined in any one of aspects 1-20 or 41, or a pharmaceutically acceptable derivative thereof, as an active ingredient for the manufacture of an anticoagulant medicament.

54. A method of treating a condition in which inhibition of thrombin is required, said method comprising administering to a human suffering from or susceptible to such a condition a therapeutically effective amount of a compound as defined in any one of aspects 1-20 or 41, or Pharmaceutically acceptable derivatives.

55. A method of treating a disorder that is an indication for anticoagulant therapy, said method comprising administering a therapeutically effective amount of a drug as defined in any one of aspects 1-20 or 41 to a human suffering from or susceptible to such a disorder. compounds or their pharmaceutically acceptable derivatives.

56. The method as described in aspect 54 or aspect 55, wherein the disorder is thrombosis.

57. The method as described in aspect 54 or aspect 55, wherein the condition is hypercoagulability in blood and/or tissue.

Claims (19)

1. formula Iization contains the pharmaceutically acceptable acid additive salt of thing,

Wherein

R ¹The C that representative is replaced by one or more fluoro substituents _1-2Alkyl;

R ²Represent C _1-2Alkyl; And

N represents 0,1 or 2.

2. the acid salt of claim 1, wherein said acid is sulfonic acid.

3. claim 1 or 2 acid salt, wherein said acid is ethyl sulfonic acid, positive propanesulfonic acid, Phenylsulfonic acid, 1,5-naphthalene disulfonic acid or normal butane sulfonic acid.

4. each acid salt of claim 1-3, wherein R ¹Representative-OCHF ₂Or-OCH ₂CH ₂F.

5. each acid salt of claim 1-4, wherein R ²Represent methylidene.

6. each acid salt of claim 1-5, wherein n represents 0 or 2.

7. each acid salt of claim 1-6, wherein said formula I compound is Ph (3-Cl) (5-OCHF ₂The CH of)-(R) (OH) C (O)-(S) Aze-Pab (OMe) or Ph (3-Cl) (5-OCHF ₂The CH of)-(R) (OH) C (O)-(S) Aze-Pab (2,6-two F) (OMe).

8. each acid salt of claim 1-7, wherein said salt are crystalline forms basically.

9. each acid salt of claim 1-7, wherein said salt is the partial crystallization form.

10. each acid salt of claim 1-8, wherein n represents 0, and described salt is crystalline form basically.

11. the acid salt of claim 1-7 each or claim 9, wherein n represents 2, and described salt is the partial crystallization form.

12. the acid salt of claim 1-8 each or claim 10, wherein said salt is Ph (3-Cl) (5-OCHF ₂The CH of)-(R) (OH) C (O)-(S) Aze-Pab (OMe) benzene sulfonate is characterized in that the feature of its X-ray powder diffraction style is to have 5.9,4.73, the peak of the d-value of 4.09 and 4.08 .

13. claim 1-7 each, the acid salt of claim 9 or claim 11, wherein said salt is Ph (3-Cl) (5-OCHF ₂The CH of)-(R) (OH) C (O)-(S}Aze-Pab (2,6-two F) (OMe) half-1, and the 5-napadisilate is characterized in that, the feature of its X-ray powder diffraction style is to have 18.3,9.1 5.6,5.5,4.13, the peak of the d-value of 4.02,3.86,3.69 and 3.63 .

14. each the method for acid salt of preparation claim 1-13, described method comprise acid is added in the formula I compound of claim 1.

15. the method for preparing acid salt of claim 14, described method comprise each acid salt of crystallization claim 1-13.

16. pharmaceutical preparation, described preparation comprise claim 1-13 each acid salt and with the pharmaceutically acceptable auxiliary of its blended, diluent or carrier.

17. be used for the treatment of each the acid salt of claim 1-13 of the illness that wherein needs Trombin inhibiting.

18. each acid salt of claim 1-13 is used for the treatment of application in the medicine of the illness that wherein needs Trombin inhibiting as active ingredient in preparation.

19. treatment wherein needs the method for illness of Trombin inhibiting, described method comprises to each the acid salt of claim 1-13 of suffering from or be easy to suffer from people's administering therapeutic significant quantity of such illness.

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